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Botanical   Microtechnique 


A    HAND.BO. 


METHODS    FOR    THE    PREPARATION,    ST^^^IfTNG, 

A^   MICROSCOPICAL   INVESTIJuAt'lON 

y'' OE-VEGETABLE    STRUC^T^ES 


/ 


!;•> 


BY 


Dr 

PRIVAT-DOCENT 


{fi, 


T    IN   t^CyNlVERaSrV    AT 


TUBINGEN 


\^   E 


JAi 


^/?(9^    r//£    GERMAN  BY 

ELLIS  HUMPHREY,  S.D. 


NEW  YORK 
HENRY  HOLT  AND  COMPANY 

1893 


Copyright,  1893, 

BT 

HENRY  HOLT  &  CO. 


ROBERT  DRDMMOND,  RLBCTROTTPER  AND  PRINTER,  NEW  TORS 


in 


PREFACE. 


The  methods  brought  together  in  the  present  volume 
are,  of  course,  chiefly  taken  from  the  Hterature  scattered 
through  various  original  papers  and  text-books.  But  the 
author  has  always  endeavored,  so  far  as  possible,  to  reach 
an  opinion  from  his  own  experience  concerning  the  methods 
described  ;  and  many  of  the  details  and  modifications  of 
previous  methods  contained  in  the  book  are  due  to  his  own 
investigations. 

However,  the  literature  used  has  been  as  fully  quoted  as 
possible  at  all  points,  in  so  far  as  it  seems  of  present  value. 
Works  of  merely  historical  interest  are  not  referred  to,  since 
the  book  is  designed  only  for  practical  use. 

If  the  writer  has  overlooked  many  statements  of  value,  it 
is  to  be  hoped  that  it  will  be  understood  and  pardoned  by 
those  familiar  with  the  immense  extent  of  botanical  litera- 
ture, especially  in  recent  years.  The  author  will  be  grateful 
to  any  one  who  will  call  his  attention  to  such  omissions. 

Regarding  the  quotation  of  literature,  it  may  be  said  that 
numerals  are  placed  after  authors'  names  in  the  text,  which 
refer  to  the  literature  hst  at  p.  v,  the  first  (Roman)  num- 
ber indicating  the  work,  and  the  second,  the  page  of  the 
work  cited.  Where  the  author  was  not  able  to  consult  the 
original  in  the  preparation  of  this  book,  the  abstracts  used 
are  referred  to. 

In  the  arrangement  of  the  organic  compounds,  Beilstein's 


5486:/ 


IV  PREFACE. 

Handbuch  der  organischcn  Chemie  (II.  Auflage,  Leipzig, 
1 886-1 890)  has  been  substantially  followed. 

The  illustrations,  where  the  contrary  is  not  expressly 
stated,  are  prepared  from  the  author's  original  drawings. 

The  manuscript  was  practically  completed  in  July,  1891  ; 
but  I  have  tried  to  include  the  more  recent  literature,  so  far 
as  possible,  during  the  printing. 

TuEBiNGPiN,   March,   1892. 


TRANSLATOR'S    NOTE. 


The  need  of  a  good  handbook  of  microscopical  methods, 
as  appHed  to  plants,  has  for  some  time  been  evident. 

Such  as  we  have  had  have  only  partially  covered  the 
ground,  and  are  now  mostly  out  of  date  on  account  of  the 
rapid  advances  of  the  last  few  years. 

The  appearance  of  the  original  edition  of  this  work  very 
satisfactorily  met  this  growing  demand  so  far  as  concerned 
students  familiar  with  the  German  language  ;  while  its  evi- 
dent thoroughness  and  the  familiarity  with  its  subject-mat- 
ter shown  by  the  author  in  the  selection  of  the  most  useful 
among  the  innumerable  published  methods  give  it  especial 
value  for  students  of  less  experience. 

The  belief  that  elementary  students  should  have  access, 
from  the  first,  to  the  best  methods,  and  the  fact  that  few 
such  English-speaking  students  read  German  readily,  have 
led  me,  with  the  support  of  the  present  pubhshers,  and  with 
the  cordial  consent  of  the  author  and  publishers  of  the  orig- 
inal edition,  to  undertake  its  translation. 

In  preparing  this  English  edition,  I  have  followed  quite 
closely  the  original.  Certain  notes  and  tables  have  been 
added  which,  it  is  hoped,  will  add  to  its  practical  usefulness 
to  American  and  English  students ;  and  certain  matters  not 
included  by  the  author,  which  seem  to  demand  notice,  have 
been  discussed  in  their  proper  places. 

V 


VI 


TRANSLATOR'S  NOTE. 


I  am  especially  indebted  to  Dr.  Zimmermann  for  prepar- 
ing, for  this  edition,  notes  on  several  important  results  of 
very  recent  studies  ;  and  I  have  added  a  few  annotations  of 
the  same  sort,  thus  bringing  the  present  edition  as  com- 
pletely as  possible  up  to  date.  All  additions  by  the  trans- 
lator are  enclosed  in  square  brackets. 

Weymouth  Heights,  Mass  ,  July,  1893. 


CONTENTS. 


PART  FIRST.     GENERAL  METHODS. 

PAGE 

1.  The  Observation  of  Living  Plants  and  Tissues.     §§  1-5 i 

2.  The  Investigation  of  Dried  Plants.     §§  6,  7 5 

3.  Maceration.     ^§  8,  9 6 

4.  Swelling.     §  10 8 

5    Clearing.     §§11-27 8 

A.  Chemical  Clearing- methods.     §  12 9 

B.  Physical  Clearing-methods.     §§  13-27 11 

1.  The  Ordinary  Method  of  Transfer  from  Water  to  Canada 

Balsam.     §§  14-22 12 

II.  The    Transfer    from    Water    to    Canada    Balsam  without 

Alcohol.     §§  23-25         17 

III.    The  Use  of   other   strongly   refractive    Mounting    Media 

§§  26,  27 18 

6.  Live  Staining.     §  28 19 

7.  Fixing  and  Staining  Methods.     §§  29-40 2a 

A.  Fixing.     §§  32-34 21 

B.  Removal  of  Fixing  Fluids.     §  35 22 

C.  Staining.      §§  36-39 24 

D.  Fixing  and  Staining  Microscopically  Small  Objects.     §  40  .     .  27 

8.  Microtome  Technique.     §§  41-52 29 

I.   Imbedding  in  Paraffine.     §§  43-49 31 

la.   Imbedding  in  Celloidin.     §  4ga 35 

II.  The  Attachment  of  Sections.     §§  50-52 37 

9.  Making  Permanent  Preparations.     §§  53-62 40 


PART  SECOND.     MICROCHEMISTRY. 
A.  Inorganic  Compounds. 

1.  Oxygen.     §  63 44 

2.  Peroxide  of  Hydrogen.     §§  64-67 45 

3.  Sulphur.     §§  68-70 47 

4.  Hydrochloric  Acid  and  its  Salts.     §  71 48 

vii 


VUl  CONTENTS. 

PACK 

5.  Sulphuric  Acid  and  its  Salts.     §  72. 49 

6.  Nitric  Acid  and  its  Salts.     §§  73-76 50 

7.  Phosphoric  Acid  and  its  Salts.     §  77 52 

8.  Silicic  Acid  and  the  Silicates.     §§  7S-81 53 

9.  Potassium,     §  82 56 

10.  Sodium.     §  83 56 

11.  Ammonium.     §  84 57 

12.  Calcium.     §§  85-99 57 

a.  Calcium  Oxalate,     gg  86-89 57 

b.  Calcium  Carbonate.     §§  90-92 60 

c.  Calcium  Sulphate.     §§  93,  94 62 

d.  Calcium  Tartrate.     §  95 63 

e.  Calcium  Malate.     §  95a 64 

/.  Calcium   Phosphate.     §§  96,  97 64, 

g.   Recognition  of  Calcium  in  the  Ash.     §98 66! 

//.   Recognition  of  Calcium  in  the  Cell-sap.     ii  99 66j 

13.  Magnesium.     §§  100,  loi 67] 

14.  Iron.     §  102 6J 

B.  Organic  Compounds. 

I.   Fatty  Series. 

1.  Alcohols.     §  103  .     , 

Dulcile.     §  103 69 

2.  Acids.     §§  104-106 701 

a.  Oxalic  Acid.     §  104 ...  701 

b.  Tartaric  Acid.     §  105 70! 

c.  Betuloretic  Acid.     §  106 .70 

3.  Fats  and  Fatty  Oils.     §§107-112 71 

4.  Wax.     §§  113-115 74 

5.  Carbohydrates.     §§  116-125 75 

a.  Glucose.     §§  1 18-120 77 

b.  Cane-sugar.     §  121 78 

c.  Inulin.     §§  122,  123 78 

d.  Glycogen.     §  124 80 

e.  Dextrine.     §  125 So 

•6.  Sulphur  Compounds.     §§  126,  127 81 

a.  Garlic-oil.     §  126 81 

b.  Mustard-oils.     §  127 81 

7.  Amido-compounds.     §§  128-130 82 

a.  Leucin.  §  129 82 

b.  Asparagin.     §  130 

II.    Aromatic     Series. 

I.  Phenols.     §§  131-133 84! 

a.   Eugenol.     §  131 84I 

If.  Phloroglucin,     ^132 84I 

c.  Asaron.     §  133 85I 


CONTENTS.  IX 

PAGE 

•2.  Acids.     §§  134-136 85 

a.  Tyrosin.     §§  134,  I35 85 

b.  Ellagic  Acid.     §  136 86 

3.  Aldehydes.     §  I37 86 

Vanillin.     §  137 86 

4.  Quinones.     §§  138-141 87 

a.  Juglon.     §  139 87 

b.  Emodin.     §  140 87 

c.  Chrysophanic  Acid.     §  141 88 

5.  Hydrocarbons.     §§  142-149 88 

a.  Ethereal  Oils.     §   144 89 

b.  Resins  and  Terpenes.     §§  145-149 90 

6.  Glucosides.     §§  150-164 92 

a.  Coniferin.      §  151 ...  92 

b.  Datiscin.     §  152 93 

c.  Frangulin.     §153 93 

d.  Hesperidin.     §  154 93 

e.  Coffee-tannin.     §  155 .  94 

/.   Potassium  Myronate,     §  156 95 

^.   Phloridzin.     §  157 95 

h.   Ruberythric  Acid.     §  158 95 

i.   Rutin.     §  159 96 

k.   Saffron-yellow.      §  160 96 

/.   Salicin.     §   i6oa 96 

m.   Saponin.     §  161 96 

n,  Solanin.     §  162 97 

o.  Syringin.     §  163 98 

/.   Glucoside  (?)  from  the  Stimulus-conducting  Tissue   of  Mimosa 

pudica.     §  164 98 

7.  Bitter  Principles.     §§  165,  166 99 

a.  Calycin.     §  165 99 

b.  SperguHn.     §  166 99 

S.  Coloring  Matters.     ^§  167-197    .     .     .      .   • 100 

a.  Pigments  of  the  Chromatophores.     §§168-179 100 

a.  Chlorophyll-green.     §  169 loi 

ft.  Carotin.     §§  170-172 loi 

y,   Xanthin.      §173 103 

d.   Coloring  Matter  of  Aloe  Flowers.     §  174 103 

6.   Coloring  Matters  of  the  /"A7;'/«V^  (Phycoerythrin).     §175.  103 

C.   Coloring  Matters  of  the /'/^^^//^/r^^(Phycophcein).   §176  .  104 

7/.  Coloring  Matters  of  the  Q/^/;/6'//zjV^^^  (Phycocyanin).   §177.  104 

0.   Coloring  Matters  of  the  Z>za^^/«rt!<:^<?  (Diatomin).     §178     .  105 
I.    Coloring  Matters  of  the   Peridineae  (Peridinin  and  Phyco- 

pyrrin).     §  179 105 

b.  Fatty  Pigments  or  Lipochromes.     §§  iSo,  181 106 

c.  Other  Coloring  Matters    dissolved   in    Fats    or   Ethereal    Oils. 

§  182 , 107 

d.  Coloring  Matters  dissolved  in  the  Cell-sap.     §§  183-185    .     .     .107 

a.   Anthocyanin.     §  184 107 


CONTENTS.  ^^^^^ 

PAGE 

y5.  Anthochlorin.     §  185 loS 

r.   Coloring  Matters  which  are  first  contained  in  the  Cell-contents, 

but  later  penetrate  the  Wall.     §§   186-188 108- 

/.  Coloring  Matters  which  only  occur  deposited  in  the    Cell- wall, 

§§  189-191 ^"9 

^.  Coloring    Matters    which    are    deposited  upon    the   Cell-wall, 

§192-197 11^ 

a.  Thelephoric  Acid.     §194 i^^ 

^.  Xanthotrametin.     §  195 113 

y.   Pigment  oi  j4^arirus  armil/a^us.     §196 113 

d.   PlgmGutoi  Paxillus  atrotomentosus.     §197 IIS 

9.  Tannins.     §^  198-208 114 

10.  Alkaloids.     §§  209-222a 119 

a.  Aconitine.     §  210 120 

b.  Atropine,     §  211 120 

c.  Berberin.     §  212 120 

d.  Brucine.     §  213 122 

e.  Colchicine.     §  214 122 

/.   Corydaline.     §  215 122 

g.  Cytisine.     §  216 123, 

h.  Opium  Alkaloids  (Morphine,  Narcotine,  NarceYne).     §  217  .     .  123 

i.   Piperine.     §  217a 124 

k.  Sinapine.     §  218 125 

/,  Strychnine.     §  219 125 

m.  Theobromine.     §  220 126 

n.  Coffeine,  Theine.     §  221 127 

o.  Veratrine.     §  222 127 

/.  Xanthine.     §  222a 128 

11.  Nitrogenous  Bases.     §  223 12& 

Nicotine.     §  223 128 

12.  The  Proteids  and  Related  Compounds.     §§  224-239 128 

a.  Reactions  of  the  Proteids.     §^  224a-234 129 

b.  Nucleins.     §§  235,  236 .     .  133 

c.  Plastin.     §  237 134 

d.  Cytoplastin,  Chloroplastin,  Metaxin,    Pyrenin,    Amphipyrenin, 

Chromatin,  Linin,  and  Paralinin.     §§  238,  239 135 

13.  Ferments.     §§  240,  241 136 

a.  Emulsin.     §  240a 136 

b.  Myrosin,     §  241 137 


PART  THIRD.     METHODS  FOR  THE  INVESTIGATION  OF   THE 
CELL-WALL  AND  OF  THE  VARIOUS  CELL-CONTENTS. 

A,  The  Cfll-wall, 

In  General.     §§  242,  243 138 

1.  The  Cellulose- wall,     §§244-250 139 

2.  Lignified  Membranes,     §§  251-261 143 

3.  The  Cuticle  and  Suberized  Membranes.     §§  262-272 148 


CONTENTS.  xi 


PAGE 


4.  Gelatinized  Cell-walls,  Plant-mucilages,  and  Gums.     §§  273-282  .     .   154 

a.  Amyloid.      §  276 1^5 

b.  Wound-gum.     §  277 icy 

c.  The  Gelatinous  Sheaths  of  the  Conjugatce.     §§  278-282     .     .     .157 

5.  Fungus  Cellulose.     §§  283,  284 160 

6.  Paragalactan-like  Substances.     §§  285-287 i5i 

a.   Reserve  Cellulose.     §  286 1^2 

I).   Paragalactan.     §  287 162 

c.  Arabanoxylan.     §  287a 163 

7.  Callus  and  Callose.     §§  288-291 163 

8.  Pectic  Substances.     §§  292-296 166 

g.  Ash-  and  Silica-skeletons  of  the  Cell-wall.     §  297 168 

ID.  On  the  Developmental  History  of  the  Cell-wall.     §  297  a-e     .     .     .   168 
II.   The  Finer  Structure  of  Cell-walls,     §  297  f-p 170 

B.  The  Protoplasm  and  Cell-sap. 

In  General.     §  298 174 

I.  The  Nucleus  and  its  Constituents.     §§  299-348 175 

I.  The  various  Methods,  in  General.     §^  300-330 176 

a.  Fixing  Methods.     §§  300-313 176 

b.  Staining  Methods.     §§  314-327 180 

c.  Simultaneous  Fixing  and  Staining.     §§  328,  329    ....  18S 

d.  Staining  intra  vitam.     §  330 189 

II.  The  Resting  Nucleus  and  its  Constituents.     §§  331-339  •     •     .  190 

a.  Its  Recognition.     §§  331-334 190 

b.  Its  Constituents.     §§  335-339 191 

III.  The  Karyokinetic  Figures.     §§  340-342 192 

IV.  The  Inclusions  of  the  Nucleus.     §§  343-348 194 

^.   The  Centrospheres.     §  348  a-d 198 

3.  The  Chromatophores  and  their  Inclusions.     §§  349-364 201 

I.  Methods  of  Investigation.     §§  350-354 201 

11.  The  Finer  Structure  of  the  Chromatophores.     §§  355-357    •     •   203 
III.  The  Inclusions  of  the  Chromatophores.     §§  358-364  ....  204 

a.  Protein  Crystalloids.     §  359 205 

b.  Leucosomes.     §  360 205 

c.  Pyrenoids.     §§  361-363 206 

d.  Oil-drops.     §  364 20S 

4.  The  Eye-spot.   §  365 209 

5.  The  Elaioplasts  and  Oil-bodies.     §§  366-370 209 

6.  The  Iridescent  Plates  of  various  Marine  Algae.     §§371-372     .     .     .   212 

7.  Microsomes  and  Granula.     §§  373-375 212 

8.  The  Cilia.     §§  376-380 214 

9.  Protein-grains.     §§  381-392 215 

a.  The  Fundamental  Mass.     §§  382-384 216 

b.  The  Crystalloids.     §§  385-387 217 

c.  The  Globoids.     §§  388-391 219 

d.  Crystals.     §  392 221 

10.  Protein  Crystalloids.     §§  393-395 222 


xu 


CONTENTS. 


PAtiK 

11.  Rhabdoids  (Plastoids).     §396 223 

12.  The  Acanthospheres  of  the  Characea.     §§  397-399 224 

13.  Starch-grains  and  Related  Bodies.     §§400-415 225 

a.  Starch.     §§  400-410 225 

b.  Floridean  Starch.     §411 230 

c.  Phaeophycean  Starch.     §  412 230 

d.  Paramylum.     §  413 230 

e.  Cellulin-grains.     §414 231 

/.   Fibrosin-bodies.     §415 231 

14.  The  Mucus-globules  of  the  Cyanophycece,     §§  416-417 232 

15.  Tannin-vesicles.     §§418-421 234 

16.  The  Reaction  of  the  Various  Cell-constituents.     §§  422-427     .     .     .  236 

17.  Plasmolysis  (Plasma-membranes).     §§  428-434 238 

18.  Methods  of  determining  whether  Certain    Bodies  lie   in  the  Cyto- 

plasm or  in  the  Cell-sap.     §§  435,  436 24a 

19.  Aggregation.     §§  437-440 241 

20.  Artificial  Precipitates.     §§  441-444 242 

21.  The  Loew-Bokorny  Reagent  for  "Active  Albumen."     §§  445-44S    .  244 

22.  Protoplasmic  Connections.     §§  449-454 245 

23.  Contents  of  Sieve-tubes.     §§  455,  456 248 

APPENDIX.     METHODS    OF    INVESTIGATION    FOR    BACTERIA. 

In  General.     §457 250- 

I.  The  Observation  of  Living  Bacteria.     §  458 25a 

II.  Fixing  Methods.     §§459-465 251 

1.  Cover-glass  Preparations.     §§  459-464 251 

2.  Sections.     §  465 253 

III.  Staining  Methods.     §§466-476 253 

1.  Loeffler's  Methylene  blue 253 

2.  Ziel's  Carbol-fuchsin 254 

3.  Ehrlich's   Solutions 254 

4.  Gram's  Method 255 

5.  Staining   Tubercle  Bacilli 256 

6.  Staining   Spores 257 

7.  Staining  Cilia 257 

Tables  for  Reference 259 

Literature  List 265 

Index 28s 


BOTANICAL   MICROTECHNIQUE. 


part  fivBt 

GENERAL  METHODS. 

I.  The  Observation  of  Living-  Plants  and  Parts  of 

Plants. 

1.  The  direct  observation  of  microscopically  small  Algae 
and  Fungi  generally  offers  no  difficulties,  and  is  best  con- 
ducted in  the  culture  fluid  of  the  organism  under  observa- 
tion. 

In  case  of  delicate  objects  which  would  suffer  from  the 
pressure  of  the  cover-glass,  this  may  be  prevented  by  the 
interposition  of  paper  strips,  capillary  tubes  of  glass,  or 
similar  objects.  The  method  proposed  by  Kirchner  (I,  VIl) 
and  Vosseler  (I,  461)  is  especially  adapted  to  this  purpose. 
It  consists  in  providing  the  cover-glass  with  small  "  wax 
feet "  at  its  corners,  for  which  a  mixture  of  wax  and  turpen- 
tine is  best.  This  is  prepared  by  adding  to  melted  wax 
one  half  or  one  third  of  its  bulk  of  Venetian  turpentine, 
while  stirring  constantly. 

This  mass  adheres  very  closely  to  glass  and  possesses 
besides  a  certain  plasticity,  so  that  one  can  readily  use  im- 
mersion lenses  and  can  easily  sHde  or  depress  the  cover- 
glass. 

2.  Where  investigations  are  to  be  continued  through  a 
longer  period  of  time,  covering  the   organisms   in  a  small 


BOTANICAL   MICROTECHNIQUE. 


'drop  of  fluid  with  a  cover-glass  in  most  cases  greatly 
hinders  the  access  of  oxygen.  In  such  cases  the  so-called 
moist-chamber  is  commonly  used.  This  is  closed  above  by 
a  cover-glass,  from  whose  lower  surface  a  drop  of  culture 
fluid,  containing  the  micro-organisms  to  be  observed,  hangs 
free  (observation  in  the  hanging  drop). 

Such  a  moist-chamber  can  be  prepared  most  simply  from 
card-board  about  2  mm.  thick.     A  rectangular  piece  is  cut 
"  from  this,  a  little  smaller 

than  the  glass  slide  to  be 
used,  and  a  square  open- 
ing is  then  cut  in  its  mid- 
dle, with  sides  about  4 
mm.  shorter  than  the 
cover-glass  to  be  used 
(cf.  Fig.  I,  a).  These 
card-board  cells  are 
thrown,  before  use,  into 
boiling  water,  by  which 
^  they   are  at   the   same 

Fig.  I.— Moist-chamber  for  the  culture  of  micro-     .  i  i  •« 

organisms.  time  Saturated  and  steril- 

ized. While  still  wet  they  are  placed  on  slides,  and  then 
the  cover-glasses,  with  the  organisms  to  be  cultivated  in 
hanging  drops  on  their  under  sides,  are  placed  upon  them. 
The  covers  are  so  placed  as  to  rest  upon  the  card-board  on 
all  sides,  as  is  shown  in  Fig.  i,  b,  which  represents  such  a 
moist-chamber  in  longitudinal  section. 

The  culture  drop  is  thus  freely  in  contact  with  a  large 
volume  of  air,  and  is  protected  from  evaporation  by  keep- 
ing the  card-board  wet  by  the  occasional  addition  of  a  few 
drops  of  water. 

Besides  this,  various  other  moist-chambers  have  been 
used  for  the  same  purpose,  as,  for  example,  slides  with 
ground  cavities,  or  glass  blocks  with  hemispherical  or  lens- 
shaped  hollows.  These  offer,  in  some  cases,  certain  advan- 
tages ;  but  in  general  they  are  based  on  the  same  principle, 
and  their  use  is  easily  understood  (cf.  Strasburger,  1,415 
ff.,  and  Behrens,  I,  51  and  162). 


GENERAL   METHODS.  3 

Moreover  it  is  usually  necessary  to  renew  the  culture 
fluid  in  all  these  moist-chambers  from  time  to  time,  perhaps 
twice  a  day.  With  some  larger  objects  this  can  be  easily 
accomplished  by  taking  up  the  drop  with  filter-paper  and 
replacing  it  by  a  fresh  one. 

3.  In  many  cases  one  may  employ  with  good  results  the 
methods  recently  recommended  by  several  writers,  which 
permit  a  continuous  change  of  the  culture  fluid.  I  will  de- 
scribe in  detail  only  the  method  of  Klercker  (II),  which 
seems  to  possess  certain  positive  advantages  over  those  of 
Rhumbler  (I)  and  Schonfeld  (I). 

J.  af  Klercker  uses  in  the  first  place  an  English  slide 
(cf.  Fig.  2)  to  which  two  glass  strips,  Z,  about  .14  mm. 
thick,  are   cemented  with   Canada  balsam,  as  shown  in  the 


Outlet. 


X^^'-' 


J-^' 


Inlet. 


Fig.  2.— Slide  for  culture  in  running  water.     After  J.  af  Klercker. 

figure.  In  the  middle  of  the  channel  thus  formed  is  placed 
the  organism  to  be  cultivated,  and  a  large  cover-glass  is  laid 
over  the  whole.  If  the  capillary  space  thus  formed  between 
the  slide,  the  two  glass  slips,  and  the  cover  is  not  wholly  filled 
with  fluid,  a  sufficient  quantity  to  fill  it  is  added.  Then  a 
strip  of  linen  is  pushed  under  the  cover  from  each  side  (5, 
Fig.  2),  and  the  latter  is  fastened  to  the  slide  with  rubber 
bands,  G.  The  slide  thus  prepared  is  attached  to  a  second 
slide  with  wax,  in  order  to  be  more  easily  movable,  and 
then  the  whole  is  fastened  to  the  stage  of  the  microscope 
with  two  clips,  K^  in  the  manner  shown  in  Fig.  3. 

The  supply  of  water  passes  through  the  siphon,  //"(Fig. 
3),  from  the  larger  beaker  (^,),  which  is  protected  from  dust 
by  a  glass  plate,  by  the  aid  of  the  linen  strip  (5,)  drawn 
through  it,  whose  free  end  lies  upon  the  linen  strip  5  (Fig. 


4  BOTANICAL   MICROTECHNIQUE. 

2.)  The  escape  is  provided  for  in  the  same  way  by  a  Hnen 
strip  (5„)  which  communicates  with  the  small  beaker  (^„). 
The  rate  of  flow  can,  obviously,  be  regulated  by  changing 
the  height  of  the  water  in  the  beaker  B, ,  as  well  as  by  fill- 
ing the  siphon  H  more  or  less  tightly  with  linen.      J.  ai 


Fig.  3.— Part  of  a  microscope  with  apparatus  for  culture  in  flowing  water.     After 
J,  af  Klercker. 

Klercker  commonly  allows   a  flow  of  about    50   ccm.  in  24 
hours. 

4.  In  order  to  protect  long-continued  cultures  of  Algae 
from  Bacteria  and  other  fungi,  one  may,  with  Klebs  (III, 
492),  add  to  the  culture  fluid  .05^  of  neutral  potassium 
chromate  (K^CrO,),  which  does  no  perceptible  injury  to 
Algae  or  to  sections  of  higher  plants.  Palla  (I,  322) 
added  for  the  same   purpose  .o\%  of   potassium   bichromate 

(K,Cr,0,)- 

5.  If  one  wishes  to  observe  sections  of  larger  plant-tissues 
in  the  living  condition,  they  must,  naturally,  be  always  more 
than  a  single  cell-layer  in  thickness,  so  that  they  may  con- 
tain cells  in  no  way  injured  by  cutting.  Most  cells,  how- 
ever, die  pretty  quickly  in  pure  water,  when  the  chromato- 
phores  and  the  nucleus  often  become  completely  deformed 
by  swelling  strongly. 

Therefore  very  various  fluids  have  been  used  for  the  study 
of  living  cells,  such  as  solutions  of  neutral  salts,  2  to  10  per 
cent  solutions  of  sugar,  gum  arabic,  and  fresh  egg-albumen. 
In  the  study  of  nuclear  divisions  in  the  embryo-sac,  a  1.4^ 


GENERAL   METHODS.  5. 

solution  of  potassium  nitrate  (saltpeter)  rendered  Treub 
(I,  9)  good  service. 

The  observation  of  uninjured  chromatophores  can  be 
very  successfully  conducted  in  many  cases,  as  Bredow  (I)  has 
stated  and  the  author  can  confirm,  by  placing  the  fresh 
sections  directly  in  oil  (pretty  fresh  olive-oil).  The  cells- 
not  only  remain  alive  in  this  for  a  long  time,  but  the  oil 
acts  as  a  clearing  agent,  from  its  strongly  refractive  prop- 
erty, and  usually  excludes  the  air  from  the  intercellular 
spaces  pretty  completely. 

When  using  other  fluids  for  observation,  however,  one 
must  commonly  use  a  filter-pump  for  removing  the  air  ;  and 
sections  on  the  slide  can  be  treated,  or  larger  pieces  of 
tissue  can  be  filled  with  fluid  before  cutting.  The  pieces 
are  placed  in  small  crystallizing  dishes  and  are  attached  by 
filter-paper  to  their  bottoms  so  as  to  remain  wholly  covered 
with  fluid  during  the  pumping. 

The  same  object  can  be  attained  by  placing  the  sections 
containing  air  in  boiled  water ;  but  in  general  the  pump- 
will  accomplish  the  desired  end  sooner. 

II.  The  Investigation  of  Dried  Plants. 

6.  For  the  investigation  of  dried  Algce  and  Fungi,  Lager- 
heim  (I  and  II)  recommends  that  they  be  first  softened  in 
water  and  then  placed  in  concentrated  lactic  acid,  in  which 
they  are  to  be  heated  until  they  show  small  bubbles.  The 
organisms  thus  treated,  having  completely  resumed  their 
original  forms,  can  then  be  directly  studied  in  the  lactic  acid. 

7.  Herbarium  material  of  the  higher  plants  may  often  be 
made  suitable  for  sections  by  simply  soaking  in  water.  In 
many  cases  the  dried  parts  may  be  treated  with  dilute  am- 
monia or  caustic-potash  solution,  which  should  be  washed 
out  before  cutting.  The  strength  of  the  solutions  and  the 
time  of  their  action  must  be  governed  by  the  nature  of  the 
objects,  and  must  be  determined  for  each  separate  case^ 
Small  or  friable  objects  are  best  cut  with  the  microtome 
after  being  imbedded  in  paraffine  or  similar  substance  (cf^ 


O  BOTA.VICAL   MICROTECHNIQUE. 

§  41  ff.).     Objects  which  have    become  strongly  colored   in 
drying  may  best  be  bleached  by  emi  de  Javelle  (cf.  §  12,  4). 

III.  Maceration. 

8.  In  many  cases,  especially  when  concerned  with  the 
size,  form,  or  structure  of  the  membrane  of  the  various 
cells,  it  is  desirable  to  separate  an  organ  into  its  compo- 
nent cells.  This  proceeding,  which  is  commonly  known  as 
maceration^  depends  upon  the  fact  that  the  middle  lamella 
which  is  present  between  adjoining  cells  is  dissolved  by 
various  reagents,  so  that  the  cells  separate  from  each  other. 
The  ready  solubility  of  the  middle  lamella  depends,  accord- 
ing to  the  researches  of  Mangin  (IV  and  VI),  in  most  cases 
upon  the  fact  that  it  consists  of  various  pectic  compounds. 
Thus  one  can  bring  about  an  isolation  of  the  cells  in  very 
many  objects  by  treating  them  first  with  acid  alcohol  and  then 
with  ammonia  (cf.  §  295). 

Besides  these,  one  may  use  many  other  and  varied  media 
for  the  same  purpose.  Thus  it  is  sufficient  in  many  cases  to 
place  the  tissues  for  a  time  in  boiling  water  or  dilute  acid,  to 
completely  isolate  the  separate  cells.  This  may  be  accom- 
pHshed,  according  to  Solla  (I),  in  juicy  fruits  by  oxalic  acid 
or  tartaric  acid,  in  potatoes  and  carrots  by  acetic  acid.  The 
endosperm  of  Phytelephas  is,  moreover,  separated  into  its 
cells  by  chlorine-water  or  caustic  potash  in  a  few  days,  or  by 
hydrochloric  acid  in  two  minutes. 

In  general,  however,  more  energetic  reagents  are  neces- 
sary for  the  isolation  of  the  separate  cells,  or  at  least  pro- 
duce that  result  more  quickly  and  certainly. 

9.  The  following  macerating  agents  are  especially  adapted 
to  more  general  use. 

^  I.  Schulzes  Maceration  Mixture  (HNO3  ana  KCIO,). — 
This  is  still  most  frequently  used  for  maceration.  It  is  best 
used  by  putting  small  pieces  or  slivers  of  the  organ  to  be 
treated  into  a  test-tube  containing  about  2  ccm.  of  ordinary 
concentrated  nitric  acid,  adding  some  crystals  of  potassium 
chlorate,  and  then  warming  the  test-tube  until  bubbles  are 


GENERAL    METHODS.  J 

freely  evolved.  The  reagent  is  then  generally  allowed  to- 
act  for  a  few  minutes  until  the  pieces  are  quite  white,  when 
the  whole  contents  of  the  test-tube  are  poured  out  into  a 
crystallizing  dish  filled  with  water.  The  pieces  of  macerated 
tissue  are  then  placed  directly,  or,  better,  after  washing  in 
pure  water,  or  also  in  alcohol,  upon  a  glass  slide.  Here 
they  can  be  readily  separated,  by  needles  or  similar  means^ 
into  their  separate  cells. 

Often  the  isolation  of  the  cells  of  large  pieces  of  tissue 
which  have  been  treated  with  the  macerating  fluid  may  be 
effected  by  shaking  the  pieces  violently  in  a  glass  half  full 
of  water. 

It  should  be  remarked  that  the  heating  of  this  macerating 
mixture  should  preferably  be  carried  on  under  a  hood,  or  at 
least  not  in  the  neighborhood  of  a  microscope,  on  account 
of  the  evolution  of  injurious  gases. 

2.  Chromic  Acid  (CrOg). — Chromic  acid  is  especially  use- 
ful for  the  isolation  of  the  cells  of  sections.  These  are 
placed  in  a  concentrated  aqueous  solution  of  the  acid,  and,, 
after  it  has  acted  for  half  a  minute  to  five  minutes,  are 
washed  in  a  large  quantity  of  water.  The  sections  are  usu- 
ally then  readily  separated  into  their  cells.  Chromic  acid 
attacks  the  cell-membranes  much  more  strongly,  when  act- 
ing longer,  than  does  Schulze's  mixture,  which  is  in  general 
preferable,  although  its  manipulation  requires  somewhat 
more  care. 

3.  Caustic  Potash. — Caustic  potash  is  useful,  especially  with 
delicate  tissues,  such  as  the  roots  of  Taraxacum  officinale. 
The  tissues  should  be  boiled  for  a  few  minutes  in  a  solution 
containing  about  ^0%  of  potassium  hydrate  and  then  placed 
in  water.  The  cells  are  then  readily  isolated  by  teasing. 
Dilute  caustic  potash  is  also  recommended  by  Solla  (I)  for 
the  isolation  of  cork  cells. 

4.  Glycerine  and  Sulphuric  Acid. — According  to  the 
method  proposed  by  A.  Fischer  (IV,  p.  xcvi),  it  is  possi- 
ble at  the  same  time  to  recognize  starch  in  the  isolated 
cells.  This  author  places  isolated  vascular  bundles  or  suit- 
able sections  in  a  solution  of  iodine  in  glycerine   under  a 


^  BOTANICAL   MICROTECHNIQUE, 

cover-glass,  places  at  the  edge  of  the  latter  a  drop  of  sul- 
phuric acid,  and  warms  the  whole  until  it  steams,  for  not 
over  a  minute.  By  pressure  on  the  cover-glass  a  complete 
isolation  of  the  separate  cells  may  be  accomplished,  and  any 
starch  present,  not  being  dissolved  by  the  sulphuric  acid 
•diluted  with  glycerine,  becomes  readily  visible  by  being 
colored  by  the  iodine.  In  the  case  of  soft  parts  of  plants, 
leaves  and  herbaceous  stems,  this  method  may  do  excellent 
service.  But  for  wood  and  the  like  I  have  not  found  it 
suited. 

IV.  Swelling. 

10.  Especially  to  bring  out  better  certain  structural  rela- 
tions of  membranes  and  starch-grains,  there  may  sometimes 
be  used  the  so-called  swelling  media,  which  produce  an 
increase  of  volume  depending  chiefly  on  increased  water- 
content. 

The  most  used  medium  is  aqueous  caustic  potash,  which 
•causes  a  greater  or  less  swelling  according  to  its  concentra- 
tion. It  is,  moreover,  very  well  adapted  for  the  study  of 
the  swelling  phenomena  of  protein  crystalloids. 

Concentrated  sulphuric  acid  is  also  a  strong  swelling 
medium,  and  finally  quite  dissolves  membranes  consisting 
of  pure  cellulose.     Cuprammonia  (cf.  §  246)  acts  similarly. 

As  a  swelling  medium  for  starch-grains  chromic  acid  has 
been  frequently  recommended. 

Finally,  Dippel  (I)  used  a  solution  of  mercuric  iodide  in  a 
potassium  iodide  solution  for  making  clear  certain  membrane 
structures.  The  proper  concentration  of  this  solution  must 
be  determined  for  each  special  case. 

V.  Clearing. 

11.  In  many  cases,  where  one  wishes  not  so  much  to  study 
the  entire  contents  of  various  cells  as  to  determine  their 
general  arrangement,  the  courses  of  vascular  bundles,  or  the 
distribution  of  less  soluble  cell-contents,  as,  for  example, 
calcium  oxalate  crystals  or  similar  bodies,  it  may  be  desira- 


GENERAL   METHODS.  9 

ble  to  make  considerable  masses  of  tissue  as  transparent 
as  possible.  For  this  clearing  of  preparations,  vegetable 
anatomists  have  heretofore  used  chiefly  strong  dissolving 
or  disorganizing  reagents,  like  caustic  potash,  chloral  hydrate, 
etc.,  which  exert  a  clearing  effect  chiefly  by  the  solution  or 
swelling  of  substances  that  hinder  observation. 

Clearing  media  play  an  important  part,  especially  in  all 
stained  preparations  ;  but  here  the  above-named  reagents 
are  not  applicable,  since  they  would  destroy  most  stains. 
In  this  case  one  must  avail  himself  almost  exclusively  of  the 
methods  for  some  time  employed  by  zoologists  and  anato- 
mists, which  consist  in  placing  the  preparations  in  strongly- 
refractive  media  like  clove-oil,  Canada. balsam,  etc.  These 
clear  less  by  destroying  than  by  equalizing  the  refractive 
differences. 

We  may  thus  conveniently  distinguish  between  chemical 
and  physical  clearing,  even  though  a  perfectly  sharp  line 
cannot  be  drawn  between  the  two  processes.  Indeed  the 
reagents  that  are  primarily  chemically  active  often  have 
besides  a  clearing  effect  due  to  their  higher  refractive 
indices.  Yet  here  there  are  always  two  essentially  different 
methods  involved,  and  a  separate  consideration  of  them 
seems  to  me  justified.  I  will,  however,  remark  that  the 
physical  clearing  methods  are  in  no  way  limited  to  stained 
preparations,  and  can  be  used  with  the  best  results,  especially 
in  investigations  with  polarized  light. 

A.    CHEMICAL   CLEARING   METHODS. 

12.  Formerly  caustic  potash  was  almost  wholly  used  for 
the  chemical  clearing  of  preparations.  More  recently  vari- 
ous other  clearing  media,  especially  phenol,  chloral  hydrate, 
and  eau  de  Javelle,  hay^  been  recommended,  and  in  most 
cases  decidedly  deserve  preference.  According  to  the 
object  of  the  investigation,  one  may  use  sometimes  one  and 
again  another  medium  with  the  best  results.  As  to  the 
manner  of  using  these  reagents,  the  following  may  be  said. 

I.  Potassium  Hydrate  (KQH)  is  used  mostly  in  aqueous 


lO  BOTANICAL    MICROTECHNIQUE. 

solution  and  in  various  degrees  of  concentration.  Besides, 
solutions  of  potassium  hydrate  in  alcohol  or  in  various  mix- 
tures of  alcohol  and  water  are  recommended  for  clearing. 
For  complete  clearing  several  hours  are  often  necessary, 
sometimes  even  several  days,  though  it  can  often  be  has- 
tened by  warming.  After  the  removal  of  the  solution  and 
superficial  washing  with  water,  the  free  potassium  is  best 
neutralized  with  dilute  hydrochloric  or  acetic  acid.  If  the 
preparations  are  then  too  opaque,  they  can  be  again  treated 
with  caustic  potash  or  made  more  transparent  with  ammonia. 

The  well-washed  preparations  can  usually  be  preserved 
for  a  time  in  glycerine-gelatine  ;  but  after  a  few  years  they 
usually  become  dark  and  often  cloudy  also. 

For  staining  preparations  treated  with  caustic  potash 
Errera  (IV)  recommends  canarin,  which  is  not  decomposed 
by  that  reagent. 

2.  Phenol  {Q^flYi). — The  best  for  clearing  is  a  solution 
of  crystallized  phenol,  which  contains  only  water  enough  to 
keep  it  fluid  at  ordinary  temperatures.  This  penetrates  cut 
parts  of  plants  relatively  fast,  and  usually  makes  them  fully 
transparent  in  a  short  time.  The  clearing  may  be  markedly 
hastened  by  heating  the  objects  in  the  phenol  solution  to 
boiling  :  and  at  the  same  time  the  air  is  completely  expelled 
from  the  intercellular  systems. 

Objects  cleared  with  phenol  can,  according  to  my  experi- 
ence, be  well  preserved  in  Vosseler's  turpentine  (cf.  §  27). 

3.  Chloral  Hydrate  (CCl,.CH(OH),)  has  heretofore  been 
used  commonly  for  clearing  in  a  concentrated  aqueous  solu- 
tion. This  can  be  used  as  well  with  fresh  as  with  alcoholic 
material.  To  hasten  the  extraction  of  chlorophyll  one  may 
use  successfully  a  concentrated  alcoholic  solution  of  chloral 
hydrate.     The  reaction  may  also  be  hastened  by  warming. 

4.  Eau  de  Javelle. — A  solution  of  potassium  hypochlorite 
(KCIO)  is  known  in  pharmacy  as  Eau  de  Javelle  (Javelle 
water).  This  can  at  any  time  be  obtained  ready  for  use 
from  an  apothecary ;  but  it  may  also  be  prepared  by  adding 
to  a  concentrated  aqueous  solution  of  chloride  of  lime  a 
solution  of  potassium  oxalate,  as  long  as  a  precipitate  is 


GENERAL    METHODS.  II 

formed.     The  solution,  filtered  from  the  precipitate,  can  be 
diluted  with  water  before  use  (cf.  Strasburger,  I,  632). 

Eau  de  Javelle  acts  like  chloral  hydrate,  but  has  the  ad- 
vantage that  it  destroys  chlorophyll  much  more  quickly. 
It  may  also  be  used  to  decolorize  dried  parts  of  plants. 

B.    PHYSICAL   CLEARING   METHODS. 

13.  Physical  clearing  will  evidently  be  the  more  complete 
as  the  refractive  index  of  the  enclosing  fluid  approaches 
that  of  the  cell-membrame  or  of  those  constituents  of  the 
cell-contents  which  are  concerned.  Thus  in  many  cases 
glycerine  must  exercise  a  certain  clearing  effect,  since  the 
refractive  index  of  pure  glycerine  (1.46)  is  markedly  higher 
than  that  of  water  (1.33).  A  much  more  complete  clearing 
is,  however,  obtained  by  various  ethereal  oils,  balsams,  and 
resins.  Among  these  Canada  balsam  plays  the  chief  part 
at  present,  and  we  will  therefore  first  describe  in  detail  the 
transfer  to  this  medium.  This  requires,  when  one  has  the 
object  in  water,  a  series  of  manipulations. 

Thus,  as  Canada  balsam  is  quite  insoluble  in  water,  a 
Complete  dehydration  of  the  object  is  first  necessary.  Since 
^this  is  commonly  accomplished  with  alcohol,  with  which 
Canada  balsam  does  not  mix,  the  replacement  of  the  alcohol 
by  a  fluid  which  will  mix  with  Canada  balsam,  xylol,  clove- 
oiL  or  the  like,  is  required. 

In  this  method,  which  may  be  termed  the  normal  or  ordi- 
nary method  of  transfer  from  water  to  Canada  balsam,  three 
distinct  manipulations  are  to  be  distinguished  :  dehydration, 
replacement  of  alcohol,  and  transfer  to  the  enclosing  me- 
dium. Whenever  in  the  following  pages  transfer  to  or 
clearing  in  Canada  balsam  is  mentioned,  the  use  of  this 
method,  which  may  perhaps  seem  somewhat  complicated  to 
beginners,  is  understood.  After  the  description  of  the  de- 
tails of  this  method,  we  shall  see  that  the  same  object  can  be 
accomplished  by  various  other  methods,  whose  use  is  neces- 
sary in  some  cases ;  for  example,  when  the  nature  of  the 
stain  forbids  the  treatment  of  the  preparations  with  alcohol. 


12  BOTANICAL   MICROTECHNIQUE. 

Finally,  under  this  head,  the  use  of  some  other  strongly 
refractive  nnedia  will  be  described. 

I.  The  Ordinary  Method  of  Transfer  from  Water  to  Canada  Balsam. 
{a)  Dehydration. 

14.  Dehydration  by  alcohol  commonly  does  not  present 
the  least  difficulty.  In  case  of  microtome  sections  it  is  suffi- 
cient to  cover  them  with  alcohol  and  then  let  the  alcohol 
flow  off  ;  free-hand  sections  are  best  placed  in  small  dishes 
or  cups  with  alcohol,  and  left  for  a  longer  or  shorter  time, 
according  to  their  thickness. 

But  there  often  results  from  direct  transfer  from  water  to 
alcohol  the  shrinking  of  the  cells  or  their  collapse  from  the 
too  rapid  withdrawal  of  their  water.  Several  methods  have 
been  employed  to  prevent  this  collapse  of  the  cells,  whose 
essential  feature  lies  in  the  very  gradual  replacement  of  the 
water  by  the  alcohol. 

This  can  be  effected  by  placing  the  preparations  in  turn 
in  different  mixtures  of  water  and  alcohol,  each  of  which 
exceeds  the  previous  one  in  its  proportion  of  alcohol.  For 
instance,  one  may  prevent  collapse  by  placing  the  prepara- 
tions first  in  \o%  alcohol  and  then  in  order  in  2>o%,  $0%,  70j^, 
90j^,  and  finally  in  absolute  alcohol.  The  time  between 
the  transfers  must  depend  upon  the  thickness  of  the  tissues. 
With  delicate  objects,  as,  for  example,  unicellular  alga^, 
intervals  of  a  few  minutes  each  are  sufficient. 

In  the  case  of  filamentous  algae  the  transfer  can  be  much 
iiimplified  by  binding  them  together  with  a  thread. 

15.  Gradual  dehydration  can  be  accomplished  by  a  method 
•devised  by  J.  af  Klercker,  which  consists  in  allowing  abso- 
lute alcohol  to  flow  slowly  into  10^  alcohol  through  a  fine 
capillary  tube. 

16.  The  dehydrating  vessel*  recommended  by  Fr.  E. 
Schulze  (I)  brings  about  the  gradual  replacement  of  water 

•  This  may  be  obtained  of  Warmbrunn,  Quilitz  &  Co.,  Berlin,  C,  Rosen- 
thalerstr.,  40,  at  the  price  of  Mk.  2.75  (67  cents). 


GEXERAL   METHODS.  1 3 

by  osmotic  action  and  is  especially  adapted  for  small  objects. 
As  is  shown  in  the  accompanying  Fig.  4,  in  which  the  bell- 
shaped  cover  that  closes  the  vessel  is  not 
shown,  this  consists  chiefly  of  two  cyl- 
inders, broadened  at  the  top  and  placed 
one  within  the  other,  their  lower  ends 
being  closed  by  a  membrane  which  per- 
mits osmotic  exchange  between  water 
and  alcohol.  Schulze  recommends  for 
this  purpose  a  thin  writing-paper  known 
as  '*  Postverdruss."  "  which  is  glued  to  the 
ground  lower  edge  of  the  cylinder. 

In  the  inner  cylinder  are   placed   the  c-  ^  x.  ^ 

•^  ^  Fig.  4.— Dehj-dratine   ves- 

objects  to  be  dehydrated  in  ver>^  dilute,  ^^-  After  f.  e.  scTiuize. 
about  10^,  alcohol ;  in  the  outer  cylinder  is  placed  a  small 
quantity  of  stronger,  about  50%,  alcohol ;  and  in  the  vessel 
containing  the  cylinders  is  absolute  alcohol,  which  is  kept 
water-free  by  a  layer  of  anhydrous  copper  sulphate  on  the 
bottom  of  the  vessel.  For  complete  dehydration  a  period 
of  twenty-four  hours  is  always  sufficient.  Further,  the 
rapidity  of  the  osmotic  interchange  may  be  largely  regu- 
lated by  changes  of  the  differences  in  level  between  the 
different  fluids.  With  less  sensitive  objects  one  may  find  one 
cylinder  sufficient,  and  then  the  dehydration  can  be  accom- 
plished in  a  few  hours. 

17.  According  to  the  method  proposed  by  Overton  (I,  12), 
dehydration  may  be  conducted  by  placing  the  objects  first 
in  \o<i  glycerine.  In  this,  objects  which  have  been  fixed 
never  suffer  collapse ;  and  living  ones  may  first  be  killed  by 
osmic-acid  fumes  (cf.  §  308).  The  preparations  are  then  left 
exposed  to  the  air  without  a  cover-glass,  but  protected  from 
dust  by  a  bell-jar.  The  solution  of  glycerine  is  thus  so  con- 
centrated by  the  evaporation  of  the  water  that  finally  a 
transfer  to  alcohol  is  possible  without  any  collapse. 

*  [The  American  papers  manufactured  under  the  name  of  "  parchment- 
paper  "and  the  finer  grades  of  the  so-called  "  Overland  paper  "serve  the  pur- 
pose well.  Best  of  all  is  true  parchment;  and  chamois-skin  has  also  been 
recommended.] 


14  BOTANICAL   MICKO'J'KCHNIQUE. 


{b)   The  Replacement  of  Alcohol. 

18.  In  earlier  years  clove-oil  was  almost  exclusively  used 
for  the  replacement  of  alcohol,  and  it  is  in  many  cases  very 
well  adapted  for  that  use  on  account  of  its  complete  misci- 
bility  with  alcohol.  For  microtome  sections  fastened  on 
the  slide  it  is  sufficient  to  place  a  few  drops  of  clove-oil  on 
the  slide  after  the  removal  of  the  alcohol.  Thicker  sections, 
especially  free-hand  sections,  are  best  placed  in  a  vessel  with 
clove-oil,  in  which  they  are  left  until  they  are  completely 
transparent  and  no  longer  appear  white  on  a  dark  ground. 

y  But  clove-oil  has  the  disadvantage  of  washing  out  many 
/  stains,  and  has  been  at  present  wholly  given  up  by  many 
microscopists,  on  account  of  its  oxidizing  characteristics. 
How  far  the  other  ethereal  oils  proposed  as  substitutes  for 
it — oil  of  origanum,  oil  of  lavender,  and  others — are  free  from 
these  disadvantages  remains  to  be  discovered. 

19.  But  in  any  event  we  have  in  xylol  a  reagent  which  can 
very  well  replace  clove-oil.  With  microtome  sections  I  use 
it  now  exclusively  except  where  I  wish  to  utiHze  the  differ- 
entiating effect  of  clove-oil,  as,  for  instance,  in  Gram's 
method  (§  321). 

y    Xylol  has  only  the  disadvantage  that  it  mixes  with  alcohol 
/  less  readily  and  requires  a  more  complete  dehydration  than 
\    clove-oil.     In   consequence  of  this   one   easily   finds   milky 
\  cloudings,    and    with    thicker    sections    would    better    use, 
between  the  alcohol  and  the  xylol,  a  mixture  of  three  vol- 
umes of  xylol  and  one  volume  of  alcohol.     For  microtome 
sections  it  is  sufficient  in  most  cases,  on  the  otbqi?  hand,  to 
cover  them  with  the  ordinary  so-called  absolute  alcohol  (98^) 
and  then  to  add  xylol.     The  beginner  will  do  well  before 
the  final  enclosure  in  Canada  balsam  to  always  examine  the 
preparations  on  a  dark  ground.     If  they  appear  white  and 
opaque,  alcohol  should  be  added  again,  and  then  xylol  again, 
until  the  preparations  have  become  completely  transparent. 
I  will  remark  here  that  in  this  case,  and  in  general  when  it 
is  desired  to  bring  somewhat  large  quantities  of  fluid  upon 


GENERAL   METHODS, 


15 


Fig.  6.— Glass  bot- 
tle with  glass  rod 
on  the  stopper. 
After  W.  Behrens 
(I). 


the  slide,  one  may  use  with  satisfaction  the  bottle  shown  in 
Fig.  5,  whose  hollow  stopper  ends  in  a  glass  tube,  while  the 
upper  end  is  closed  by  a 
rubber  cap.  By  compress- 
ing the  rubber  cap  and  then 
allowing  it  to  expand,  fluid 
is  drawn  into  the  stopper, 
and  may  then  be  pressed 
out  in  suitable  quantities 
by  renewed  pressure  on 
the  rubber  cap.  With  re- 
agents which  are  to  be  used 
only  in  drops,  the  bottle 
figured  in  Fig.  6,  whose 
stopper  is  drawn  out  simply  Fig.  5  —Glass  bottle 

.     ,  ,  ,  1  with  pipette.  After 

mto    a  glass    rod,   may  be     w.  Behrens  (i). 
used. 

20.  In  order  to  prevent  the  collapse  of  delicate  objects 
when  brought  into  clove-oil  or  xylol  we  may  use  the  meth- 
ods proposed  by  Overton  (I,  12). 

I.  If  an  object  is  to  be  brought  into  clove-oil  or  other  ethe- 
real oil,  it  is  taken  from  the  alcohol  and  placed  in  a  small 
dish  containing  a  10^  solution  of  the  oil  in  alcohol.  This 
dish  is  then  placed  in  a  somewhat  larger  one  or  in  a  suitable 
exsiccator,  whose  bottom  is  covered  with  solid  calcium  chlo- 
ride. The  alcohol  is  then  gradually  absorbed  by  the  chlo- 
ride, and  the  object  becomes  at  last  completely  saturated 
with  oil.  To  prevent  longer  action  of  the  alcohol,  one  may 
transfer  the  objects  from  alcohol  to  water-free  chloroform 
and  thence  to  a  \o%  solution  of  clove-oil  in  chloroform,  from 
which,  as  in  the  last-described  method,  the  chloroform  may 
be  absorbed  by  calcium  chloride. 

II.  For  transfer  to  xylol  the  objects  are  put  in  a  dish  with 
a  \o%  solution  of  xylol  in  alcohol  and  placed  in  a  exsiccator 
on  whose  bottom  is  pure  xylol.  Such  an  adjustment  then 
takes  place  between  the  two  fluids  by  diffusion,  that  the 
objects  finally  lie  in  nearly  pure  xylol. 

21.  The  transfer  of  very  small  objects  from  alcohol  to 


i6 


BOTANICAL   MICROTECHNIQUE. 


xylol  may  also  be  accomplished  by  means  of  the  settling-cyl- 
inder^   recommended  by  Fr.    E.    Schulze  (I) 
which  makes    possible  at  the  same  time   the 
transfer  from  xylol  into  Canada  balsam.     In 
this  vessel,  whose  construction  is  evident  from 
the  accompanying  Fig.  7  without  further  ex 
planation,  are  placed  three  different  fluids  in 
layers  above  one  another.       Below   is  xylol- 
Canada  balsam,  next  xylol,  and  finally  alcohol 
In  the  latter  are  placed  the  previously  dehy 
drated  objects.     If  they  are  pretty  small  the 
sink  so  gradually  to  the  bottom  that  they  com 
^^^Aftfr  ^"^^  ^^^   Canada  balsam   without    collapsing 
By  means  of  the  cock  on  the  side  of  the  cylin 
der  the  xylol  and  alcohol  may  be  drawn  off,  and  the  prepara- 
tions may  then  be  removed  directly  to  balsam  on  the  slide* 


Fig.  7.  — 
cylinder 
Fir.  E.  Schulze, 


{c)   Transfer  into  the  Enclosing  Medium. 


22.  For  enclosing  zoological  or  botanical  preparations  in 
Canada  balsam   one  ordinarily  uses  fluid   Canada  balsam 
prepared  by  dissolving  this  resin  in  chloroform,  xylol,  o 
some  similar  solvent.     The  solution  in  xylol 
is  especially  to  be  recommended.     This  may 
be   poured  into   a  wide-mouthed   glass  (cf. 
Fig.  8),  whose   cover  fits  over   the    mouth 
outside,  and  is  so  high  that  there  is  room 
for  a  small  glass  rod  in  the  closed  vessel. t 

No  collapse  occurs  in  transferring  an  ob- 
ject from  clove-oil  or  xylol  to  this  fluid  Can- 
ada balsam,  as  a  rule.  For  very  delicate 
objects  the  ordinary  xylol-balsam  may  well 
be  diluted  with  xylol,  and  then  the  latter 
may  be  allowed  to  gradually  evaporate. 


Fig.  8. 


Canada-bal- 
sam gla&s,  about  ^J 
n<it.  bize. 


*  This  may  be  obtained  of  Warmbrunn,  Quilitz  &  Co.,  Berlin,  C,  Rosen-] 
thalerstr.,  40,  at  the  price  of  Mk.  3.25  (80  cents). 

f  Such  glasses  among  others  may  be  obtained  at  60  pf.  (15  cents)  each  of| 
Dr.  G.  Grtibler  (Leipzig,  Bayerische  Str.,  12). 


GENERAL   METHODS.  1/ 

That  collapsing  may  also  be  prevented  by  the  use  of 
Schulze's  settling-cylinder  has  already  been  noticed  (cf.  §  21). 

2.  The  Transfer   from  Water   to  Canada   Balsam  Without  the  Use 

of  Alcohol. 

{a)  By  Drying. 

23.  A  transfer  from  water  to  Canada  balsam  can  be 
accomplished  without  the  use  of  alcohol  by  simply  letting: 
the  preparations  dry  in  the  air,  then  covering  them  with 
xylol,  which  usually  penetrates  quite  dry  sections  rapidly,, 
and  then  enclosing  them  in  xylol-Canada  balsam.  Naturally 
this  method  is  applicable  only  to  such  preparations  as  suffer 
no  collapse  from  drying,  especially  to  very  thin  microtome 
sections. 

{b)   With  Aniline  {C.H^NH,). 

24.  Since  about  4^  of  water  is  soluble  in  aniline,  the  latter 
can  be  used  for  dehydration.  The  preparations  are  brought 
directly  from  water  into  aniline,  and  may  then  be  mounted 
in  Canada  balsam.  The  aniline  may  be  dehydrated  by  solid 
potassium  hydroxide  (KHO),  which  is  wholly  insoluble  in  it 
(cf.  Suchanek  I). 

{c)   With  Phenol  {C.HfiH). 

25.  If  sections  are  transferred  from  water  to  phenol  which 
has  been  melted  by  warming  in  a  paraffine  oven  (cf.  §  47)  or 
by  the  addition  of  a  little  water,  they  are  cleared  in  a  short 
time  and  sufficiently  dehydrated  to  be  transferred  directly 
to  clove-oil  or  xylol. 

To  prevent  the  collapsing  of  very  delicate  objects,  the 
method  proposed  by  Klebahn  (I,  419)  may  be  used.  The 
fixed  and  stained  objects  are  first  placed  in  dilute  glycerine, 
which  is  allowed  to  concentrate  in  the  air.  Then  phenol  is 
added,  and  clove-oil  or  creosote  is  gradually  mixed  with  it, 
when  the  objects  may  be  directly  transferred  to  Canada 
balsam.  Klebahn  used  these  methods  especially  in  the 
study  of  the  germinating  spores  of  Desmids  and  carried  on 


1 8  BOTANICAL   MICROTECHNIQUE. 

the  various  manipulations  on  slides  with  hollows  ground  in 
them. 

3.  The  Use  of  Other  Strongly  Refractive  Mounting  Media. 

{ci)  Daimnar  Lac. 

26.  Dammar  lac  is  best  dissolved  in  equal  parts  of  benzol 
and  oil  of  turpentine.  Its  use  is  the  same  as  that  of  Canada 
balsam,  from  which  it  differs  in  its  somewhat  lower  refrac- 
tive index.  Thus  differences  of  structure  that  depend  on 
differences  in  refractive  power  often  become  more  conspic- 
uous. The  glasses  described  in  §  22  may,  of  course,  be  used 
for  this  medium.  It  appears  to  have  been  employed  little 
in  botanical  microscopy. 

{b)    Venetian    Tui'pentine. 

27.  This  mounting  medium  proposed  by  Vosseler  (II)  is 
prepared  by  thinning  the  resin  obtained  from  the  apothecary 
under  this  name  with  an  equal  volume  of  alcohol  and  then 
warming  it  on  a  water-bath,  shaking  it  energetically  and 
finally  filtering.  The  filtrate  is  then  somewhat  thickened 
on  the  water-bath. 

The  fluid  so  obtained  has  the  advantage  of  mixing  with- 
out cloudiness  with  90^  alcohol,  and  thus  it  makes  prelim- 
inary clearing  with  clove-oil  unnecessary  in  cases  of  incom- 
plete dehydration.  Besides,  even  with  delicate  objects,  like 
Spirogyra,  transfer  from  alcohol  to  Venetian  turpentine  much 
less  often  causes  collapse  than  does  transfer  to  clove-oil, 
Canada  balsam,  and  the  like.  One  may  avoid  the  crumpling 
of  very  delicate  objects  by  placing  them  first  in  a  mixture 
of  10  parts  turpentine  and  100  parts  alcohol,  and  then  per- 
mitting a  gradual  concentration  of  the  turpentine  over  an- 
hydrous calcium  chloride,  according  to  the  method  of 
rfeiffer  (I,  30).  If  small  dishes  are  used  for  this  purpose, 
they  maybe  provided  with  ridges  of  paraffine,to  prevent  the 
turpentine  from  rising  on  their  sides,  by  simply  dipping 
them  to  the  proper  depth  in  melted  paraffine. 

The  refractive  index  of  Venetian  turpentine  lies  between 


GENERAL   METHODS.  1 9 

that  of  glycerine  and  that  of  dammar  lac,  so  that  cell-mem- 
branes and  starch  grains  stand  out  pretty  sharply  in  it. 

A  disadvantage  of  this  medium  lies  in  the  fact  that  it  be- 
comes solid  very  slowly.  In  order  to  secure  a  firm  attach- 
ment of  the  cover-glass  to  the  slide,  which  is  often  very  de- 
sirable in  studies  with  immersion  lenses,  one  may  apply  a 
heated  metal  wire  to  the  edge  of  the  cover-glass,  as  Vosseler 
(II,  297)  has  done.  The  same  object  may  also  be  attained, 
according  to  Pfeiffer  (I),  by  encircling  the  cover  with  Canada 
balsam. 

Various  stained  tissues,  especially  carmine,  haematoxylin, 
and  saffranin  preparations,  may  be  excellently  preserved  in 
Venetian  turpentine,  according  to  Vosseler.  In  my  own 
experience,  however,  acid  fuchsin  seems  to  be  more  poorly 
preserved  in  it  than  in  Canada  balsam. 

VI.  Staining  of  Living  Tissues. 

28.  As  has  been  shown  especially  by  the  researches  of 
Pfeffer  (II),  it  is  possible  in  very  many  cases  to  cause  living 
plants  and  parts  of  plants  to  take  up  certain  coloring  mat- 
ters. This  so-called  live  staining  is  not  only  of  great  impor- 
tance for  the  study  of  the  transportation  of  material  within 
the  vegetable  organism,  but  has  also  led  to  some  interesting 
results  concerning  the  morphology  of  the  cell,  and  should 
certainly  be  capable  of  still  wider  application. 

For  the  success  of  live  staining  it  is  of  primary  importance 
that  the  staining  solution  used  should  exercise  no  injurious 
effect  upon  the  objects  concerned.  Since  the  anihne  colors 
generally  act  as  poisons  on  plant-cells,  it  is  necessary  to  use 
them  in  very  dilute  condition,  when  they  affect  the  cell  very 
little  or,  in  general,  not  injuriously.  An  evident  staining 
can,  it  is  clear,  only  take  place  when  the  stain  is  stored  up 
by  certain  constituents  of  the  cell.  This  is  generally  the 
case  when  the  osmotic  balance  between  the  cell  fluid  and 
the  surrounding  staining  solution  is  constantly  destroyed  by 
a  chemical  metamorphosis  of  the  stain  taken  up.  But  to 
make  possible  in  this  way  the  storage  of  large  quantities  of 
staining  material,  it  is  also  necessary  to  furnish  the  objects 


20  BOTANICAL   MICROTECHNIQUE, 

a  large  quantity  of  the  staining  solution.  This  staining 
should  therefore  be  carried  on,  not  on  the  slide,  but  in 
dishes  or  beakers  which  will  contain  at  least  a  half  liter  of 
fluid ;  and  not  too  many  of  the  objects  to  be  stained  should 
be  placed  in  each.  Finally,  the  rapidity  of  the  staining  may 
be  hastened  by  agitating  the  fluid. 

VII.  Methods  of  Fixing  and  Staining. 

29.  In  the  investigation  of  the  various  plasmatic  constit-  T 
uents  of  the  protoplasm,  which  are  largely  colorless  and 
commonly  distinguished  only  by  slight  differences  in  refrac- 
tive power,  it  is  often  impossible  in  difficult  cases  to  reach 
positive  results  with  living  material.  But  there  may  be 
used  with  the  best  results  the  methods  of  fixing  and  staining 
devised  chiefly  by  anatomists  and  zoologists.  These  have 
already  led  to  such  important  results  in  the  study  of  the 
vegetable  organism  that  not  the  least  doubt  remains  as  to 
their  applicability  in  botanical  investigations.  Yet,  on  the 
other  hand,  it  cannot  at  all  be  maintained  that  the  study  of 
living  material  should  now  be  wholly  given  up.  On  the 
contrary,  it  should  be  used,  whenever  at  all  possible,  for  the 
control  and  explanation  of  the  results  obtained  from  stained 
preparations. 

30.  The  purpose  of  fixing  is  to  kill  the  object  in  such  a 
way  as  to  preserve  its  structural  relations  as  completely  as 
possible  after  the  removal  of  the  fixing  medium. 

It  is  the  object  of  staining  to  so  color  certain  particular 
cell-constituents  of  the  fixed  preparations,  which  are  to  be 
specially  studied,  that  they  shall  be  sharply  differentiated 
from  their  surroundings,  so  that  their  confusion  with  other 
cell-constituents  may  be  prevented. 

We  possess  at  present  an  innumerable  lot  of  fixing  and 
staining  methods,  and  already  the  most  various  new  or 
long-known  organic  and  inorganic  compounds  have  been 
tested  with  reference  to  their  usefulness  as  fixing  and  stain- 
ing media.  While  most  of  these  experiments  .have  led  to 
no  new  conclusions  concerning  the  morphology  of  the  cell, 
and  many  methods  warmly  recommended  by  their  discov- 


GENERAL   METHODS.  21 

erers  closely  resemble  methods  long  known,  yet  a  thorough 
trial  of  all  possible  salts,  acids,  and  coloring  matters  should 
not  be  omitted.  In  consequence  of  the  slight  insight  which 
we  have  been  able  to  obtain  into  the  mechanics  of  staining,, 
it  is  only  possible  to  discover  useful  methods  in  a  purely 
empirical  way ;  and  we  cannot  yet  conceive  how  far  newly- 
discovered  stains  or  new  methods  may  lead  toward  further 
conclusions  concerning  the  structure  of  the  protoplasmic 
constituents  of  the  cell. 

31.  The  result  of  staining  is  dependent  not  only  on  the 
nature  of  the  stain  used  and  of  its  solvent,  but  also  largely 
on  the  previous  treatment  of  the  object,  especially  on  the 
fixing  medium.  Besides,  useful  results  may  be  obtained 
after  staining  by  treatment  with  various  solutions  of  salts,. 
acids,  alkahes,  and  the  like,  or  by  the  combination  of  differ- 
ent stains. 

We  shall  become  acquainted  in  the  third  part  of  this  book 
with  a  large  number  of  methods  for  fixing  and  staining. 
But  here  only  the  general  technique  of  fixing  and  staining 
will  be  described. 

A.    FIXING. 

32.  Fixing  is  generally  the  more  complete  the  more 
rapidly  the  fixing  fluid  reaches  the  cells  to  be  fixed  ;  there- 
fore the  best  results  are  obtained  with  solutions  as  concen- 
trated as  possible,  so  far  as  they  do  not  cause  precipitates  or 
exert  any  destructive  action.  Further,  small  objects  are 
more  quickly  penetrated  by  fixing  fluids  than  larger  ones,, 
and  therefore  in  difificult  cases  the  smallest  possible  pieces,, 
even  to  sections  a  few  cells  in  thickness,  should  be  placed 
in  the  fixing  fluid. 

It  should  be  especially  observed  that  cuticle  and  cork  are 
not  easily  permeable  by  most  fixing  fluids  and  indeed  are 
quite  impermeable  by  some.  One  may,  therefore,  often 
greatly  aid  the  penetration  of  fixing  fluids  by  removing 
suberized  membranes  as  far  as  possible,  or  at  least  by  splitting 
them  to  furnish  points  of  entrance  for  the  fluids. 

From  the  above  it  follows  that,  in  objects  which  hav^e  not 


22  BOTANICAL   MICROTECHNIQUE. 

been  uniformly  acted  on  by  the  fixing  fluids,  those  parts 
^vhich  lie  nearest  the  cut  surfaces  deserve,  in  general,  the 
L;reatest  confidence  in  their  study. 

ZZ'  I^  the  objects  to  be  fixed  are  very  light,  they  can  be 
-easily  attached  by  strips  of  filter-paper  to  the  bottom  of  the 
vessel  containing  the  fixing  fluid.  Especially  with  objects 
which  are  with  difficulty  wetted,  it  is  often  useful  to  inject 
them  with  the  fixing  fluid,  which  is  easily  done  by  the  aid 
of  a  filter-pump. 

It  is  not  possible  to  give  general  statements  as  to  the 
iime  necessary  for  complete  fixing.  This  depends,  aside 
from  the  size  of  the  object,  primarily  upon  the  character  of 
the  fixing  fluid  used,  and  sufficiently  exact  statements  on 
this  point  will  be  given  in  the  description  of  the  different 
methods. 

So,  too,  the  quantity  of  fluid  to  be  used  varies  much.  In 
general,  one  needs  relatively  little  of  the  energetic  fluids, 
hke  those  containing  sublimate,  for  example ;  while,  espe- 
cially where  potassium  bichromate  is  employed,  the  use  of 
large  quantities  of  fluid  is  recommended. 

34.  For  the  fixing  of  objects  which  easily  turn  black 
Overton  (I,  9)  recommends  alcohol  containing  sulphurous 
acid.  He  prepares  this  by  adding  to  \  gram  of  sodium  sul- 
phite (NajSOg)  a  few  ccm.  of  80^  sulphuric  acid  and  con- 
ducting the  fumes  of  sulphurous  acid  which  arise,  directly 
into  100  grams  of  alcohol.  Picric  acid  dissolved  in  water  or  in 
30  to  50^  alcohol  may  be  combined  with  sulphurous  acid  in 
the  same  way.  In  preparations  treated  with  the  fluids  named, 
the  finest  protoplasmic  differentiations  are  preserved,  and 
staining  with  haemotoxylin  or  carmine  succeeds  finely. 

B.    REMOVAL   OF   FIXING    FLUIDS. 

35.  It  is  necessary,  in  general,  before  staining  to  remove 
completely  the  fixing  fluid  used.  The  fluid  to  be  used  for 
this  washing  of  the  preparation  depends  upon  the  character 
of  the  fixing  medium.  If  this  is  readily  soluble  in  water,  it 
is  best  to  use  running  water,  and  for  this  purpose  the  drain- 
ing-boxes  recommended  by  Steinach  (I)  are  well  adapted,  as 


GENERAL   METHODS. 


25 


also  for  other  uses.  These  contain,  as  Fig.  9  shows  in  sec- 
tion, a  glass  drainer  which  rests  on 
three  small  glass  feet  and  has  in  its 
bottom  many  perforations  which  be- 
come wider  downward.  The  outer 
box  serves  to  enclose  the  objects  air- 
tight, for  other  uses.* 

If  many  objects  are  to  be  washed  at 
the  same  time  in  running  water,  a  use- 
ful aid  is  the  washing  device  which  was  set  up  two  years  ago 
(1890)  in  this  botanical  institute,  and  which  has  proved  veV 
satisfactory. 

This  apparatus,  whose  construction  is  readily  understood 
from   the   accompanying   Fig.   10,  consists   essentially  of  a 
ft 


— ■'    ■■■\      limn    I |. 

J 

^'^-  9  — Steinach's  draining- 
box  in  vertical  section.  After 
Steinach  (I). 


Fig.  id.— Washing  apparatus. 

brass  tube  a  provided  with  nine  small  cocks  and  the  zinc 
vessel  d  for  the  reception  of  the  objects  to  be  washed.  But 
since  the  small  cocks  cannot  sustain  the  full  pressure  of  the 
water-pipes,  the  complete  shutting  off  and  the  approximate 
regulation  of  the  water  pressure  may  be  accomplished  by 
means  of  the  large  cock  b,  which,  by  means  of  a  T-tube,  as 

*  These  boxes  can  be  obtained  of  R.  Siebert,  Wien  VIII,  Alserstr.  19,  and 
latterly  also  of  Dr.  Griibler.  The  latter  furnishes  the  glass  drainers  alone  at 
Mk.  1.25  (31  cents)  each. 


24 


BO  TANICAL   MICRO  TECHNIQ UE. 


at  c,  can  easily  be  fitted  laterally  to  any  water-faucet.  In 
the  zinc  vessel  the  larger  space  d  serves  to  hold  the  glass 
drainers.  If  the  water  is  to  run  rapidly  from  it,  the  pinch- 
cock  g  is  opened,  so  that  the  water  runs  out  through  the 
tube  /  that  communicates  with  the  bottom  of  the  vessel. 
If  the  water  is  to  run  from  it  more  slowly,  the  pinch-cock 
g  is  closed,  and  the  water  can  then  escape  only  through  the 
tube  r,  whose  mouth  is  15  mm.  above  the  bottom  of  the 
vessel,  so  that  the  water  stands  1 5  mm.  deep  on  its  bottom. 
For  the  use  of  the  space  //,  see  §  39. 


C.  STAINING. 

36.  If  large  objects  have  been  fixed,  it  is  usual,  after 
washing,  to  cut  them  into  sections  and  to  stain  these.  In 
many  cases,  however,  very  good  results  are  obtained  by 
staining  the  objects  directly  after  washing  and  then  pre- 
paring sections  from  them.  In  case  of  this  so-called  stain- 
ing in  mass  ("  Stiickfarbung"),  the  slight  permeability  of  the 
cuticle  must  again  be  noted,  and  the  penetration  of  the  stain 
must  be  aided  by  its  entire  removal  or  by  slitting  it. 

In  many  cases  of  mass  staining  it  may  be  useful  to  obtain 
on  one  and  the  same  section  all  the  different  grades  of  stain- 
ing side  by  side,  the  parts  nearest  to  the  surfaces  being  most 
deeply  stained,  while  the  intensity  of  the  stain  gradually 
decreases  as  the  distance  from  the  surface  increases. 

If  a  large  number  of  objects  are  to  be  stained  at  the  same 
time,  one  may  conveniently  use  the  glass  drainer  described 
in  §  35.  Especially  with  smaller  parts 
or  sections  of  plants,  one  often  finds 
the  glass  vessel  shown  in  Fig.  1 1  useful. 
Its  cover  has  a  groove  corresponding  to 
the  edge  of  the  dish.* 

If  concentrated  staining  solutions  are 
used  in  this  case,  it  is  often  difficult  to 
recognize  the  separate  objects  clearly.     One  may  then  find 

*  [These  so-called  "  Slender  dishes,"  as  well  as  the  other  items  of  glass- 
ware described  in  these  pages,  may  now  be  obtained  of  the  leading  American 
dealers  in  microscopical  supplies.] 


^ 


Fio.  II.— Glass  dish  with 
cover,  in  median  [sec- 
tion. 


GENERAL   METHODS.  2$ 

convenient  a  little  apparatus  to  which  Ranvier  has  given  (I, 
66)  the  name  photophore  (light-bearer),  but  which  may  better 
be  called,  with  Obersteiner  (I,  55),  section-finder.  It  consists 
of  a  wooden  box,  about  5  cm.  high  and  10  to  12  cm.  long 
and  wide,  whose  front  side  is  wanting,  while  the  top  is 
replaced  by  a  plate  of  clear  glass.  There  is  placed  in  this 
box  a  small  mirror  so  inclined  toward  the  front  that  it  forms 
an  angle  of  25°  to  30°  wdth  the  bottom.  If  this  mirror  is 
now  turned  toward  a  brightly-lighted  window,  it  will,  plainly, 
reflect  the  light  against  the  plate  of  glass  forming  the  top  of 
the  box  and  against  the  dish  of  staining  fluid  placed  upon 
it.     One  can  easily  prepare  such  an  apparatus. 

The  apparatus  described  by  Eternod  (I,  41),  shown  in  Fig. 


Fig.  12. — Eternod's  apparatus. 

12,  is  also  very  convenient.  In  this  the  front  part  of  the 
glass  plate  r,  which  is  lighted  by  the  mirror  ^,  serves  as  a 
section-finder,  while  beneath  the  hinder  part  of  the  glass 
plate  d-d  is  a  strip  of  paper  divided  into  variously  colored 
portions.  The  outlines,  prepared  with  a  diamond,  at  g 
serve,  as  is  plain  without  further  explanation,  for  the  center- 
ing of  preparations  on  the  slide,  and  the  small  turn-table  ^, 
for  preparing  cement  rings,  etc. 

37.  Microtome  sections  which  are  to  be  stained  after  cut- 
ting are  usually  fastened  to  the  slide  (see  §§  50-52).  If 
it  is  desired  to  place  a  large  number  of  sections  in  the 
same  staining  fluid  at  the  same  time,  this  may  be  readily 
accompHshed  with  the  aid  of  a  number  of  crystallizing 
dishes  placed  inside  of  each  other,  as  shown  in  Fig.  13. 
The  space  between  two  dishes  is  then  filled  with  the  stain- 


26 


BO TANICAL   MICRO  TECHNIQ UK. 


ing  fluid,  and  the  slides  are  placed  in  it  so  that  the  sides 
bearing  the  sections  are  turned  outward.  Any  injury  to  or 
scraping  off  of  the  sections  is  thus  prevented.  It  is  best  not 
to  fasten  the  dishes  together,  but  simply  to  place  them  inside 
of  each  other  and  to  load  the  inner  one  with  shot  or  other 
weight,  to  prevent  their  being  floated  by  the  fluid  in  the 
outer  dish.  In  order  to  distinguish  the  different  objects  in 
the  apparatus  from   each  other,   we  may  use  crystallizing 


Fig.  13.— Apparatus  for  staining  microtome  sections. 


dishes  provided  with  lips  for  pouring.  If  then  the  appa- 
ratus is  always  filled  In  regular  sequence  in  the  same  direc- 
tion from  this  lip,  it  is  only  necessary  to  note  the  numerical 
positions  of  the  separate  preparations. 

38.  For  further  details  the  reader  is  referred  to  the  de- 
scription of  individual  methods.  Here  it  may  be  said  that 
preparations  are  rarely  to  be  studied  in  the  condition  in 
which  they  are  taken  from  the  staining  fluid.  Often  very 
useful  effects  are  obtained  by  first  strongly  "overstaining" 
the  preparations  and  then  washing  them  in  water,  alcohol, 
dilute  acid,  or  the  like,  until  only  certain  parts  of  the  cell 
contents  appear  colored.     In  many  cases  the  treatment  of 


GENERAL   METHODS.  2/ 

stained  preparations  with  several  different  fluids  is  necessary 
to  obtain  well-differentiated  images. 

39.  To  wash  microtome  sections  in  running  water  the 
apparatus  described  and  figured  in  §  35  may  be  conven- 
iently used  by  placing  the  slides  in  an  oblique  position  in 
the  space  //  of  the  zinc  vessel,  so  that  the  sides  bearing  the 
sections  are  directed  downward.  To  provide  for  the  con- 
stant escape  of  water  from  the  bottom  of  this  space,  the 
zinc  strip   /  is  perforated  like  a  sieve  near  its  lower  ^^^'^j 

vhile   the  somewhat    lower   strip,  k^   is    unperforated,   but 
n^es  to  prevent  the  too  rapid   fall   of   the  fluid   in   the 
:jace  //. 

D.    FIXING  AND  STAINING   MICROSCOPICALLY  SMALL 
OBJFXTS. 

40.  The    fixing   and    staining    of    microscopically   small 
^ject5  offers  certain  technical  difficulties,  especially  if  one 

has  but  a  limited  quantity  of  material.  These  are  best 
vercome  by  the  methods  devised  by  Overton  (I,  13),  which- 
^an  be  variously  modified  to  meet  the  peculiarities  of  the 
objects  concerned.  Thus  it  seems  to  me  more  cenvenient 
to  carr>'  on  the  manipulations  to  be  described  on  the  slide 
rather  than  on  the  cover-glass,  as  Overton  recommends^ 
unless  for  special  reasons  culture  in  the  hanging  drop  is^. 
necessar>'. 

In  the  first  place,  for  fixing  the  objects  in  a  drop  of  cul~ 
-:re  fluid  an  easily  removable  fixing  medium  should  be 
used.  For  this  purpose  the  fumes  of  iodine  are  well 
adapted.  They  are  poured  out  upon  the  preparation  front 
a  heated  test-tube,  and  are  easily  driven  off  again  by  subse- 
quent warming  (2  to  5  minutes  in  the  paraflfine  bath).  For 
the  same  purpose  the  fumes  of  osmic  acid  may  be  used. 
They  are  applied  by  holding  the  slide,  with  the  objects 
downward,  over  the  mouth  of  a  bottle  containing  a  dilute 
solution  of  osmic  acid.* 

*  [Where  this  ifnretskm  of  the  slide  cannot  be  safely  risked,  the  addition  to 
the  cnhure  Said  oC  a  drop  of  a  i^  solotioa  of  ocouc  acid  may  save  the  same 

^1 


28  BOTANICAL  MICROTECHNIQUE. 

After  fixing,  the  objects  are  transferred  to  alcohol.  This 
is  accomplished  by  first  adding  a  drop  of  10-20^  alcohol, 
which  causes  no  collapse,  and  then  placing  the  preparation 
in  a  close  chamber  saturated  with  alcohol  vapor,  to  bring 
about  a  gradual  concentration  of  the  alcohol.  For  this 
purpose  a  flat  crystallizing  dish  may  be  used,  its  upper  edge 
being  ground  so  as  to  be  hermetically  sealed  by  a  greased 
glass  plate.  Its  bottom  is  covered  with  absolute  alcohol 
and  a  stand  to  hold  the  preparations  is  placed  in  it.  The 
latter  may  be  made  by  simply  bending  down  the  ends  of 
some  strips  of  sheet  zinc.  According  to  Overton,  the  cover- 
glass  with  the  objects  is  placed,  with  its  wet  side  upward, 
on  a  piece  of  elder  pith,  about  3  mm.  high  and  of  a  diameter 
less  than  that  of  the  cover-glass,  which,  in  its  turn,  rests  on 
an  ordinary  slide.  In  this  apparatus,  which  must  be  pro- 
tected from  sudden  changes  of  temperature  and  especially 
from  direct  insolation,  the  20^  alcohol  on  the  cover-glass 
becomes  almost  absolute  alcohol  in  a  few  hours,  by  diffusion 
through  the  air.  When  this  is  accomplished,  a  drop  of  a 
dilute  solution  of  celloidin  is  placed  on  the  cover-glass  and 
evenly  spread  over  its  upper  surface  by  tipping  it  backward 
and  forward.  It  is  of  advantage  to  make  the  celloidin  film 
as  thin  as  possible,  since  thicker  films  both  separate  more 
easily  and  render  the  subsequent  manipulations  much  more 
difficult.  Therefore  pretty  thin  solutions  of  celloidin  must 
be  used.  A  suitable  one  may  be  prepared  by  diluting  the 
ordinary  ofificinal  solution  of  celloidin  with  ten  times  its  bulk 
of  a  mixture  of  equal  parts  of  alcohol  and  ether.* 

As  soon  as  the  celloidin  no  longer  flows  evidently,  the 
whole  cover-glass  (or  slide)  is  placed  in  80^  alcohol,  wet  side 
up.  Here  the  celloidin  film  becomes  so  hard  in  a  few 
minutes  that  the  objects  can  be  placed  in  suitable  staining 
fluids  without  being  washed  away.  A  long-continued  action 
of  alcohol  of  more  than  90%  is  to  be  avoided,  as  it  dissolves 

*  [The  thinnest  of  the  celloidin  solutions  recommended  for  use  in  imbed- 
ding (cf.  §  49a)  may  be  diluted  with  twice  its  own  bulk  of  the  alcohol-ether 
mixture  for  this  purpose.] 


GENERAL   METHODS.  29 

the  celloidin  film.  Therefore  Overton  dehydrates  with  80 
to  85  %  alcohol  and  uses  for  the  transfer  to  Canada  balsam, 
creosote,  which  will  mix  with  ev^en  70^  alcohol.  From 
the  creosote  the  preparations  are  either  placed  directly  in 
Canada  balsam  or  are  passed  first  through  xylol.  Aniline 
may  be  used  for  the  same  purpose  by  passing  the  objects 
from  90%  alcohol  to  aniline,  then  to  a  mixture  of  equal 
parts  of  aniline  and  xylol,  and  then  to  xylol  (cf.  §  24). 

It  should  be  noted  also  that  many  stains,  as,  e.g..  Gentian 
violet,  stain  the  celloidin  film  strongly  and  are,  therefore, 
not  to  be  used  with  this  method. 

VIII.  Microtome  Technique. 

41.  While  the  microtome  has  been  generally  used  for 
years  by  anatomists  and  zoologists,  it  has  been  used  by 
botanists  in  a  comprehensive  way  only  in  recent  years. 
But  since,  so  far  as  I  know,  no  one  who  has  recently  taken 
the  trouble  to  familiarize  himself  with  the  technique  of  the 
microtome,  has  denied  the  great  value  of  microtome  methods, 
it  seems  superfluous  to  discuss  here  in  detail  their  advantages 
and  disadvantages. 

I  will  only  remark  that  the  methods  described  in  the  fol- 
lowing sections  are  not  applicable  to  very  hard  objects, 
especially  to  woods,  while  they  have  given  me  excellent  re- 
sults with  all  soft  structures  and  also  with  most  leaves  and 
herbaceous  stems  or  roots. 

How  far  the  methods  proposed  by  Vinassa  (I  and  II)  for 
cutting  very  hard  objects,  aided  by  a  firmer  microtome, 
specially  constructed  for  this  purpose,  are  capable  of  general 
application,  I  cannot  judge  for  want  of  personal  experience. 
At  all  events  it  would  be  desirable,  where  possible,  to  so 
modify  Vinassa's  methods  as  to  preserve  the  protoplasmic 
elements  in  the  preliminary  preparation  of  objects.* 


*  [The  Providence  microtome,  devised  and  sold  by  Rev.  J.  D.  King,  Cot- 
tage City,  Mass.,  is  especially  constructed  for  cutting  hard  objects  and  is  said 
to  be  well  adapted  to  its  purpose,  but  I  am  not  able  to  speak  from  expe- 
rience of  it.] 


30  BOTANICAL  MICROTECHNIQUE. 

42.  I  refrain  from  describing  here  in  detail  the  various 
microtomes  and  their  manipulation,  and  merely  remark  that 
I  have  obtained  the  best  results  with  a  relatively  small 
microtome  by  Schanze,  namely,  sections  of  i  micron  and 
even  of  fractions  of  a  micron  in  thickness. 

In  this  instrument,  as  may  be  seen  in  Fig.  14,  the  move- 
ment of  the  knife,  as  well  as  the  raising  of  the  object,  is 
accomplished  by  the  aid   of  screws.     To  obtain  very  thin 


Fig.  14.— Microtome  by  Schanze. 

sections,  one  turns  the  disk  which  is  connected  with  the 
object-raising  screw  by  a  system  of  cogs.  This  shows  di- 
rectly I  }x  of  thickness  and  permits  the  estimation  of  frac- 
tions of  that  thickness.* 

I  have  also  worked  for  a  long  time  with  a  more  elaborate 
microtome  by  Aug.  Becker  (Gottingen)  which  was  very 
exactly  constructed. 

[1  have  used  with  great  satisfaction  for  serial  sections  of 
objects  imbedded  in  parafifine  the  Minot  microtome.  This 
has  the  knife  fixed,  while  the  object  is  moved  vertically  past 
its  edge,  being  pushed  forward  by  an  amount  equal  to  the 
desired  thickness  of  a  section,  at  each  descent,  by  an  auto- 
matic device.     The  operation  of  the  instrument  consists  in 


♦  This  microtome  was  constructed  from  the  specifications  of  Prof .  Altmantr 
and  may  be  had  of  the  mechanician  M.  Schanze  (Leipzig,  Brtiderstr.  63.) 


GENERAL   METHODS.  3 1 

the  rotation  of  the  balance-wheel  by  hand  power  or  by  a 
water  motor,  and  its  work  is  thus  more  uniform  and  more 
rapid  than  that  of  the  sledge  , 

microtomes  of  the  Schanze  and    \^^^  ^.-  ^^f^mmmmiis^^ 

,^,  _,    .  ,         .,      ,      1="-----    --^--    -     ."iPiiiilWiiiiiiiiiiiniiiiiiiiiiniiiiiiiiiiiiniiiiniini 

Thoma  types.     It  is  not  suited    y"^        .    .  -y^^"*— i— ^mjp' 
for  cutting    objects    in    celloi-     .„^        ^     ^ 

o  J  Fig.  15.— Microtome  knife.     After 

^in.*l  Henking. 

Of  the  many  microtome  knives  used  I  have  found  most 
suitable  that  recommended  by  Henking  (I)  with  a  very  short 
€dget  (cf.  Fig.  15). 

I  must  particularly  describe  the  imbedding  of  objects  to 
be  cut  and  the  manipulation  of  microtome  sections,  espe- 
cially their  attachment  to  the  slides.  But  it  cannot  be  my 
duty  to  bring  together  the  very  numerous  methods  recom- 
mended by  various  authors.  It  will  be  better  for  me  to 
confine  myself  to  the  careful  description  of  a  few  methods 
whose  trustworthiness  I  have  had  opportunity  to  prove. 
Therefore  I  will  particularly  describe,  among  the  various 
modes  of  imbedding,  only  the  paraffine  method,  which  is  by 
far  the  best  adapted  to  vegetable  objects. 

I.  Imbedding  in  Paraffine. 

43.  For  imbedding  in  paraffine,  objects  stained  in  mass  or 
unstained  objects  may  be  used.  If  one  is  concerned  with 
protoplasmic  structures,  these  must,  of  course,  be  carefully 
fixed  and  the  fixing  medium  must  be  washed  out  before 
imbedding. 

The  size  of  the  pieces  to  be  imbedded  depends  naturally 
upon  the  nature  of  the  object.  In  general  it  is  advantageous 
to  use  as  small  pieces  as  possible,  for,  on  one  hand,  these 
are  more  easily  penetrated  by  the  various  fluids,  and,  on  the 

*  [This  microtome  is  sold  by  the  Franklin  Educational  Co.,  Hamilton  PI., 
Boston,  at  $60,  with  knife.] 

f  These  are  to  be  obtained  of  W.  Walb  (Heidelberg,  Hauptstr.  5)  under 
the  name  of  "  Henking's  microtome  knife,"  at  the  price  of  Mk.  4.50  ($i.io) 
«each. 


32  BOTANICAL   MICROTECHNIQUE. 

other  hand,  it  is  the  easier  to  obtain  very  thin  sections  the 
smaller  the  surface  to  be  cut  is. 

44.  It  is  also  in  no  respect  unimportant  what  sort  of  paraf- 
fine  is  used.  I  have  used  in  most  cases  a  parafifine  recom- 
mended by  Altmann  which  melts  at  58°  to  60°  C*  and  is 
obtained  from  the  drug-store  of  Franz  Wittig  (Leipzig).  But 
when  the  size  of  the  sections  is  more  important  than  their 
extreme  thinness,  as  may  be  the  case  where  one  wishes  a 
general  view,  paraffine  to  which  has  been  added  more  or  less 
of  the  superheated  paraffine  recommended  by  Count  Spee  is 
more  useful.  Objects  imbedded  in  this  have  the  advantage 
of  rolling  up  much  less  easily  during  cutting. 

This  superheated  paraffine  may  be  prepared  by  heating 
ordinary  paraffine  in  an  open  dish  for  one  to  six  hours  until 
it  has  assumed  a  brownish-yellow  color  like  that  of  yellow 
wax,  with  the  evolution  of  disagreeable  white  fumes,  a  slight 
reduction  of  its  volume,  and  the  elevation  of  its  melting 
point.  Recently  such  superheated  parafifine  can  be  ob- 
tained directly  from  Dr.  G.  Griiblerf  and  others. 

45.  In  all  cases  a  complete  dehydration  must  precede  the 
transfer  to  parafifine.  This  can  ordinarily  be  accompHshed 
by  means  of  alcohol.  Delicate  objects  are  better  not  trans- 
ferred directly  from  water  to  alcohol,  in  order  to  avoid  col- 
lapse ;  but  one  of  the  methods  for  dehydration  described  in 
§§  14  to  17  may  be  used.  In  general  it  is  sufificient  to  use 
between  water  and  alcohol  a  mixture  of  equal  parts  of  both 
fluids,  in  which  the  objects  are  left  for  an  hour  or  longer. 
Afterwards  they  are  left  in  absolute  alcohol  from  six  to 
twenty-four  hours  according  to  their  size  ;  or  even  several 
days,  in  some  cases. 

46.  From  alcohol  the  objects  are  passed  to  a  mixture  of 


*  [If  paraffine  of  just  this  melting  point  cannot  be  obtained,  it  may  be 
readily  prepared  by  mixing  two  paraffines  of  respectively  higher  and  lower 
melting  points  in  proper  proportions.] 

f  [Dr.  Grtiblcr's  stains,  mounting  media,  and  other  preparations,  which 
are  of  standard  excellence,  may  now  be  obtained  of  Eimer  &  Amend,  Third- 
Avenue,  New  York,  and  of  the  Franklin  Educational  Co.,  Boston.] 


GENERAL   METHODS.  33 

three  parts  by  volume  of  xylol  to  one  part  of  alcohol,  in 
which  they  remain  twelve  to  twenty-four  hours.*  From 
this  they  are  placed  in  xylol  for  twelve  to  twenty-four  hours 
more.  Their  complete  permeation  with  xylol  may  be  recog- 
nized by  the  preparations  becoming  transparent. 

I  may  here  remark  that  chloroform,  oil  of  turpentine,  and  • 
toluol  have  been  used  instead  of  xylol  in  this  transfer 
from  alcohol  to  paraffine.  But  I  think  it  doubtful  whether 
these  substances  offer  any  advantage  ov^er  xylol.  How- 
ever, the  above-mentioned  mixture  of  alcohol  and  xylol  is 
decidedly  preferable  to  the  clove-oil  formerly  used  between 
the  alcohol  and  xylol. 

47.  From  xylol  the  objects  are  transferred  to  melted 
paraffine  ;  but  to  prevent  the  collapse  which  almost  always 
occurs  on  a  direct  transfer  from  xylol  to  paraffine,  it  is  better 
to  interpolate  a  solution  of  paraffine  in  xylol.  I  ordinarily 
proceed  in  the  following  manner  with  the  best  results.  I 
place  in  a  so-called  bird's  trough\  a  mixture  of  xylol  and 
paraffine  which  is  solid,  or  at  least  of  a  thick  consistency,  at 
ordinary  temperatures  ;  an  exact  statement  of  proportions  is 
not  important  here.  On  this  cold  and  soHd  paraffine-xylol 
mixture  I  place  the  objects  to  be  cut  and  pour  over  them 
enough  pure  xylol  to  cover  them.  Then  I  place  the  dish 
uncovered  on  the  top  of  the  paraffine  oven  about  to  be 
described,  where  the  mixture  melts  gradually  so  that  the 
objects  covered  with  xylol  can  sink  into  it.  A  further  con- 
centration of  the  xylol-paraffine  is  brought  about  by  the  eva- 
poration of  the  xylol.  After  six  to  twenty-four  hours  I  place 
the  objects  in  a  porcelain  dish  filled  with  melted  paraffine, 
which  I  place  in  the  paraffine  oven,  A^ile  I  allow  the  dish 
with  the  xylol-paraffine  mixture  to  cool  for  future  use  in 
the  same  way. 

The  objects  remain  in  a  dish  full  of  melted  paraffine  from 
twelve  to  twenty-four  hours  according  to  their  size,  but  a 
longer  time  is  seldom  necessary. 

*  For  all  these  transfers  Sleinach's  glass  drainers  are  very  useful, 
f  [Any  small  porcelain  dish  will  serve.] 


34 


BO  TANICA  L   MICRO  TECHNIQ  UE. 


Fig.  i6.— Paraffine  oven. 


For  ?i.  faraffine  cn>en  I  use  in  Tubingen  with  the  best  results 

tan    ordinary   double  -  walled    drying- 
I  oven    (cf.    Fig.    i6)  with    the    mantle 

filled  with  liquid  paraffine,  into  which 
^iH^B     projects  a  Desaga  thermostat.     This 
^^L   is  so  arranged  that   the  temperature 
''  f^B     ^^  ^^^  ^u\d  paraffine  is  about  63°  C* 

f^^  48.  Finally,  to  enclose  the  object 
quite  saturated  with  paraffine  in  a 
block  of  paraffine  suitable  for  cutting, 
it  is  convenient  to  place  a  drop  of 
glycerine  in  a  watch-glass  of  about 
60  mm.  diameter  and  to  rub  it  over 
the  inner  surface  of  the  glass  until  the 
glycerine  can  no  longer  be  seen.f 

The  waich-glass  is  then  somewhat  warmed  and  filled 
with  melted  paraffine.  When  this  has  cooled  to  near  its 
melting-point,  which  may  be  known  by  its  hardening  at  the 
edges  when  lightly  blown  upon,  the  objects  to  be  enclosed 
are  placed  in  it  and  so  oriented  with  a  heated  needle  that 
suitable  blocks  of  paraffine  can  be  cut  from  the  mass  when 
-cool. 

In  order  to  prevent  crystallization  in  the  cooling  paraffine 
•so  far  as  may  be,  it  should  be  cooled  as  rapidly  as  possible. 
This  is  best  accomplished  by  placing  the  watch-glass,  as 
soon  as  the  objects  are  oriented,  upon  a  large  vessel  of  cold 
Avater,  where  it  will  readily  float  if  carefully  placed  upon 
the  surface.  For  the  same  reason  it  is  desirable  to  place 
the  objects  near  the  edge  of  the  watch-glass  where  the  par- 
affine is  thinnest.  When  the  paraffine  is  quite  cold  it  sepa- 
rates easily  from  the  watch-glass  and  is  then  cut  into  rect- 
angular blocks  two  or  three  centimetres  long,  each  of  which 


♦  The  so-called  '•  Naples  waier-bath  "  recommended  by  Paul  Mayer  (I)  is 
also  very  convenient.  [This  bath  in  various  more  or  less  modified  forms 
may  be  obtained  of  American  dealers  and  in  its  best  forms  is  very  useful  in 
the  work  of  imbedding  and  mounting.] 

f  This  serves  the  purpose  of  aiding  the  subsequent  separation  of  the  paraf- 
fme  from  the  watch-glass. 


GENERAL   METHODS.  35 

lias  near  one  end  an  object  to  be  cut.  The  opposite  end  is 
to  be  put  into  the  object-carrier  of  the  microtome.  But 
lirst  the  end  containing  the  object  is  to  be  so  trimmed 
down  that,  while  the  object  is  still  wholly  enclosed  in  paraf- 
iine,  the  surface  to  be  cut  shall  be  rectangular  and  as  small 
as  possible.  In  cutting,  this  rectangle  should  be  so  placed 
that  two  of  its  sides  are  parallel  to  the  edge  of  the  micro- 
tome knife,  which  is  placed  at  right  angles  to  the  direction 
of  its  motion. 

49.  It  may  be  observed  here  that  small  blocks  of  paraffine 
can  be  easily  attached  to  rectangular  blocks  of  cork,  which 
may  then  be  fixed  in  the  object-carrier  of  the  microtome. 
It  is  only  necessary  to  place  a  few  drops  of  melted  paraffine 
on  one  face  of  the  cork  and  then  to  quickly  put  the  paraffine 
block  upon  it  and,  by  running  around  its  edges  a  heated 
metal  instrument,  to  cause  it  to  be  completely  attached  to 
the  block.  This  proceeding  is  especially  convenient  if  one 
wishes  to  change  abruptly  the  direction  of  sections,  as  from 
transverse  to  longitudinal.  If  it  is  desirable  to  cut  off  the 
paraffine  block  for  this  purpose,  it  may  be  done  with  a  knife 
heated  over  a  flame,  as  in  this  way  the  crumbling  of  the 
paraffine  is  prevented. 

For  preserving  blocks  of  paraffine,  the  boxes  used  for  the 
so-called  Swedish  matches  are  convenient;  [or  any  small 
pasteboard  boxes.] 

la.  Imbedding  in  Celloidin. 

[49a.  While  the  above-described  imbedding  method  and 
medium  are  unquestionably  of  the  first  value  in  both  animal 
and  vegetable  histology,  the  use  of  celloidin  as  an  imbedding 
medium  has  recently  become  so  extended,  and  the  service 
it  renders  in  many  cases  where  paraffine  does  not  do  well, 
Is  so  good  that  some  account  of  the  manner  of  its  employ- 
ment should  be  given  here.  It  has  the  advantage  that  no 
heat  is  required  in  the  process  of  imbedding,  and  that  very 
large  sections  may  be  cut  ;  and  the  disadvantage  that  the 
knife  must  be  kept  wet  while  cutting,  and  that  the  thinnest 


5G  ^^^FiffAT^TCAL   MICROTECHNIQUE.  ^^H 

sections  which  can  be  cut  from  it  are  relatively  thick  as 
compared  with  those  which  may  be  cut  from  paraffine. 

Objects  to  be  imbedded  in  celloidin  must  first  be  thor- 
oughly dehydrated,  preferably  in  Schultze's  apparatus  (cf. 
§  i6).  They  are  placed  in  a  mixture  of  equal  parts  of  abso- 
lute alcohol  and  sulphuric  ether,  and  then,  after  a  few  hours, 
in  a  solution  of  celloidin  in  the  above  mixture,  which  should 
contain,  according  to  Busse  (I),  one  part  by  weight  of  cel- 
loidin to  15  parts  of  the  solvent.  After  it  is  thoroughly 
penetrated  by  this  solution,  which  will  require  from  a  few 
hours  to  a  few  days,  according  to  its  size  and  nature,  the 
object  passes  to  a  stronger  solution  containing  one  part  of 
celloidin  in  1 1  parts  of  the  solvent ;  and  finally,  after  well 
penetrated  by  this,  to  a  still  stronger  one  with  the  propor- 
tion of  one  to  eight  parts.  After  remaining  for  a  suitable 
time  in  the  last  solution,  the  object  is  ready  for  imbed- 
ding. For  this  purpose,  a  paper  strip  may  be  wound 
tightly  about  the  end  of  a  small  block  of  suitable  size  and 
material,  preferably  of  bass-wood  or  of  vulcanized  fibre, 
so  as  to  form  the  sides  of  a  box  whose  bottom  is  the 
end  of  the  block.  This  box  is  now  filled  with  the  thick- 
est solution  of  celloidin,  and  in  it  the  object  is  placed 
and  oriented  carefully  by  means  of  needles  wet  with  the 
ether-alcohol  mixture.  As  soon  as  the  solvent  has  evap- 
orated sufficiently  to  form  a  firm  film  over  the  surface  of 
the  mass,  the  whole  may  be  immersed  in  alcohol,  where  it 
becomes  quite  hard  in  a  few  hours.  Since  very  strong 
alcohol  dissolves  celloidin,  it  cannot  be  used  ;  and  statements 
vary  widely  as  to  the  best  strength  for  this  purpose.  Busse 
(II)  has  found,  however,  that  85^  alcohol  gives  the  best 
results,  both  as  regards  the  transparency  of  the  celloidin 
and  the  thinness  of  the  sections  which  may  be  cut  from  it. 

The  paper  is  removed  from  about  the  celloidin  mass, 
after  it  has  hardened,  leaving  it  attached  to  the  block. 
The  mass  is  now  trimmed  to  present  a  rectangular  upper 
face,  and  the  block  clamped  upon  the  microtome  so  that  the 
object  may  be  cut  in  the  desired  plane. 

To   cut  successful   sections   from   a   celloidin   block,  it  is 


GENERAL   METHODS.  37- 

necessary  to  set  the  knife  very  slightly  oblique,  and  as 
nearly  as  possible  parallel,  to  the  direction  of  its  motion,  so 
that  the  celloidin  shall  be  cut  with  a  long  drawing  stroke. 
The  knife  and  the  top  of  the  block  should  also  be  kept  wet,, 
during  cutting,  with  alcohol  of  the  strength  of  that  in  which 
the  block  was  hardened.  With  these  precautions  excellent 
sections  may  be  obtained. 

Busse  (I)  recommends  the  use  of  photoxylin  instead  of 
celloidin,  as  it  gives  a  more  completely  transparent  imbed- 
ding mass.  The  details  of  its  manipulation  are  precisely 
the  same  as  for  celloidin.  More  detailed  accounts  of  the 
use  of  celloidin  may  be  found  in  papers  by  Eyclesheimer 
(I)  and  Koch  (I).] 

2.  The  Attachment  of  Sections. 

50.  For  the  purpose  of  dissolving  out  the  paraffine  fron> 
microtome  sections  filled  with  it,  these  are  commonly  at- 
tached to  the  slide.  Although  recently  a  large  number  of 
methods  for  accomplishing  this  have  been  proposed,  I  will 
restrict  myself  to  describing  somewhat  in  detail  four  of 
them,  each  of  which  seems  to  possess  certain  advantages 
for  some  cases. 

A.   ATTACHMENT  WITH   COLLODION. 

In  the  first  of  these  a  solution  of  about  5^  of  officinal 
collodion*  is  used  for  attachment.  It  is  conveniently  kept 
in  a  bottle  having  a  soft  brush  inserted  through  its  cork.  A 
drop  of  this  solution  is  first  allowed  to  flow  under  the  sec- 
tions arranged  as  desired  on  a  sHde,  a  piece  of  filter-paper 
is  then  laid  upon  them,  and  the  sections  are  pressed  down 
upon  the  slide  with  the  finger  or  with  a  paper-knife  or  simi- 
lar instrument.  Then  the  sections  are  painted  over  with 
the  collodion  solution  and  it  is  allowed  to  dry  in  the  air. 
When  this  is  done,  the  slide  is  warmed  over  a  small  flame 

*  [Or  a  mixture  of  equal  parts  of  a  thin  solution  of  collodion  and  clove-oil. 1 


3« 


BO  TA NICA L   MICRO  TECHNIQ UE. 


until  the  paraffine  melts,  and  then  plunged  in  xylol  t^fff 
solve  the  paraffine. 

After  this  the  sections,  if  from  objects  stained  in  mass, 
can  be  at  once  enclosed  in  xylol-Canada  balsam.  But  if 
they  are  to  be  stained,  they  must  first  be  brought  into  water 
or  alcohol  according  to  the  nature  of  the  stain  to  be  used. 
Since  the  separation  of  the  sections  from  the  slide  has  often 
occurred  during  this  transfer,  I  now  perform  it  by  carrying 
the  preparations  from  xylol  successively  into  a  mixture  of 
three  parts  xylol  and  one  part  alcohol,  90^  alcohol  and  50^ 
^.Icohol,  leaving  them  in  each  fluid  two  minutes,  or  as  much 
longer  as  is  necessary. 

I  use  for  this  purpose  vessels  with  parallel  sides,  on  the 
bottom  of  each  of  which,  at  one  of  the  short  sides,  a  piece  of 
cork  about  i  cm.  high  has  been  fastened.  The  slides  are 
then  so  placed  in  these  that  one  end  rests  on  the  piece  of 
cork  and  the  side  bearing  the  sections  is  turned  downward. 

From  50^  alcohol  the  preparations  can  be  transferred  to 
water  or  any  suitable  staining  fluid,  without  fear  of  the 
separation  of  the  sections.  At  least,  I  have  experienced 
such  a  result  very  rarely  even  in  the  use  of  the  most  com- 
plicated staining  processes  when  the  above  precautions  have 
been  observed  ;  and  I  have  not  been  troubled  by  any  seri- 
ous staining  of  the  delicate  collodion  film  by  any  of  the 
more  important  methods. 

[50a.  Cclloidin  sections,  when  arranged  on  the  slide,  may 
be  attached  to  it  by  placing  the  whole  in  a  close  chamber 
over  ether.  The  ether  vapor  quickly  dissolves  the  celloidin 
sufficiently  to  cause  the  sections  to  adhere  firmly  to  the 
slide  on  removal  from  the  chamber.  Should  any  difficulty 
be  experienced,  the  sections  may  be  arranged  on  a  thin 
collodion  or  celloidin  film  on  the  slide  and  then  treated  as 
above.  After  they  are  attached,  they  may  be  stained  and 
mounted  as  described  for  paraffine  sections  (§  50).  Objects 
stained  in  mass  may  be  imbedded  in  cellodin  as  well  as  in 
paraffine. 

For  mounting  in  Canada  balsam,  celloidin  sections  may 
be  cleared  with  a  mixture  of  three  parts  xylol  and  one  part 


GENERAL   METHODS.  3^ 

phenol,  or  with  equal  parts  of  phenol  and  oil  of  bergamot, 
or  with  oil  of  bergamot  alone.  The  last  two  are  especiall}r 
recommended.  When  objects  are  stained  before  imbedding-, 
the  whole  block  may  be  cleared  before  cutting.] 

B.    ATTACHMENT   WITH   AGAR-AGAR. 

51.  Agar-agar  is  recommended  by  Gravis  (I)  for  the 
attachment  of  microtome  sections.  A  yL^  aqueous  solu- 
tion of  this  substance  is  warmed  for  some  time  after  the 
mixture  of  the  ingredients  until  quite  homogeneous,  then 
filtered  through  a  fine  cloth  or  through  glass  wool,  and  finally 
protected  from  spoiling  by  the  addition  of  some  pieces  of 
camphor. 

A  drop  of  this  solution  is  placed  on  the  carefully  cleaned 
slide,  the  sections  are  laid  upon  this  drop,  and  the  whole  is 
warmed  until  the  paraffine  becomes  soft  without  wholly 
melting.  Crumpled  sections  then  spread  out  completely. 
After  the  cooling  of  the  slide  the  superfluous  agar-agar  is 
taken  up  with  filter-paper  and  the  rest  is  allowed  to  dr}^  com- 
pletely. After  this  the  paraffine  may  be  removed  by  xylols 
as  in  the  previous  method,  and  the  slide  may  be  transferred 
to  alcohol. 

This  method,  which  I  have  recently  tried  many  times^ 
has  the  advantage  that  it  admits  of  the  use  of  rolled  sec- 
tions ;  and  even  crumpling  due  to  the  imbedding  may  be 
wholly  or  largely  overcome.  I  have  seen  a  troublesome 
staining  of  the  agar-agar  only  with  haematoxylin. 

A  disadvantage  of  the  method,  however,  consists  in  the 
fact  that  in  pure  water  the  solution  of  agar-agar,  and  there- 
fore the  separation  of  the  sections,  often  occurs.  But  one 
can  always  treat  sections  attached  with  agar-agar  with  solu- 
tions in  strong  or  50.^  alcohol,  and  can  usually,  with  some 
care,  stain  them  with  aqueous  solutions. 

C.    COMBINED   AGAR-AGAR-COLLODION   METHOD. 

The  separation  in  water  of  sections  attached  with  agar- 
agar  can  be  prevented  by  painting  over  sections  attached  by 


40  BOTANICAL   MICROTECHNIQUE. 

the  method  just  described,  after  they  have  fully  dried,  with 
the  above-mentioned  collodion  solution  (cf.  §  $o)  and  letting 
them  dry  in  the  air.  I  have  used  this  method  often  recently, 
and  can  recommend  it  heartily  for  difficult  cases.  It  unites 
the  advantages  of  both  methods  in  that  it  makes  possible 
the  recovery  of  collapsed  sections  and  permits  the  use  of 
aqueous  stains  without  fear  of  separation  of  the  sections. 

D.    ATTACHMENT   WITH    ALBUMEN. 

52.  According  to  P.  Mayer's  (I,  II)  methods,  a  solution  of 
albumen  is  used  for  attaching  sections.  This  is  prepared 
by  mixing  50  cc.  of  the  albumen  of  hens'  eggs  with  50  cc. 
of  glycerine  and  i  gram  of  sodium  salicylate,  and  filtering 
the  mixture  after  hard  shaking.  A  small  drop  of  this  solu- 
tion, which,  according  to  Vosseler  (I,  457),  becomes  useless 
in  about  six  months,  is  placed  on  a  carefully  cleaned  slide 
and  is  rubbed  with  the  finger  or  a  soft  cloth  until  a  barely 
visible  film  remains  upon  the  slide.  The  sections  are  placed 
upon  this  and  pressed  down  upon  the  slide,  a  dry  brush 
being  held  between  the  finger  and  the  sections.  If  the 
i5lide  is  now  heated  over  a  small  flame  until  the  paraffine 
melts,  the  sections  become  so  firmly  attached  by  the  coagu- 
lation of  the  albumen  that  the  paraffine  can  be  dissolved 
out  with  xylol  or  other  solvent  without  fear  of  their  being 
washed  away.  Nor  does  this  occur  when  they  are  trans- 
ferred directly  from  xylol  to  alcohol  or  from  alcohol  to 
Avater.  Neither  have  I  observed  the  staining  of  the  albu- 
men film  by  any  coloring  matter ;  so  that  this  method  may 
be  most  conveniently  used  for  most  cases. 

IX.  Making  Permanent  Preparations. 

53.  One  may  use  very  various  methods  for  preserving 
preparations  as  long  as  possible.  In  nearly  all  cases  prepa- 
rations enclosed  in  Canada  balsam  or  some  other  resin  or 
balsam  possess  the  greatest  permanence.  But,  on  account 
of  their   high   refractive  index,  which  nearly   corresponds 


GEiXERAL    METHODS,  4 1 

with  those  of  cellulose  and  of  most  of  the  contents  of  the 
vegetable  cell,  these  substances  are  hardly  to  be  used  except 
for  stained  preparations  or  such  as  are  intended  for  observa- 
tion by  polarized  light.  Besides,  the  transfer  of  easily  col- 
lapsible objects  to  balsam  is  so  complicated  that  other 
mounting  media  are  preferable  for  them.  It  depends 
wholly  on  the  nature  of  the  objects  to  be  mounted  what 
mounting  medium  is  best  to  be  used  ,  and  it  will  be  neces- 
sary, in  the  second  and  third  parts  of  this  book,  to  re- 
peatedly indicate  what  method  of  preservation  is  best 
adapted  to  the  case  in  hand. 

Since  the  methods  of  mounting  in  Canada  balsam.  Dam- 
mar lac  and  turpentine  have  been  already  described  in 
§§  14  to  27,  only  the  remaining  methods,  in  which  glycer- 
ine especially  plays  an  important  part,  need  be  here  brought 
together. 

54.  Glycerine. —  Pure  glycerine  in  various  degrees  of  dilu- 
tion, or  a  mixture  of  this  with  an  acid,  was  formerly  a  much 
esteemed  and  almost  universally  used  medium.  In  its  use, 
however,  especial  care  must  be  taken  that  the  glycerine  used 
is  not  diluted  by  too  long  exposure  to  the  air,  since  in  that 
case  a  gradual  drying  up  of  the  preparation  takes  place,  if 
the  subsequently  applied  cement  ring  (cf.  §  62)  is  not  air- 
tight. Concentrated  glycerine  often  cannot  be  used,  how- 
ever, on  account  of  its  strong  clearing  and  dehydrating 
power.  In  such  cases  a  dilute  solution  of  glycerine  with  a 
few  drops  of  acetic  acid  offers  great  advantages,  but  must, 
as  already  remarked,  be  very  carefully  protected  against 
evaporation.     (Cf.  especially  Dippel,  II,  loio.) 

55.  Glycerine  and  Chrome  Almn. — P'or  preserving  prepara- 
tions of  Schizophycece  and  Floridece  in  their  natural  colors, 
Kirchner  uses  (I,  p.  Vll)  dilute  glycerine,  to  which  is  added 
enough  chromium-potassium  sulphate  (chrome  alum)  to  give 
the  fluid  a  clear  bluish  color. 

56.  Glycerine-gelatine. — This  is  of  late  most  used  and 
offers  undeniable  advantages,  in  most  cases,  over  the  fluid 
glycerine  mixtures.  It  is  conveniently  prepared  from  the 
recipe  recommended  by   Kaiser,  as  follows  :  One  part   by 


42  BOTANICAL   MICROTECHNIQUE. 

weight  of  gelatine  is  soaked  in  six  parts  of  water ;  seven 
parts  of  pure  glycerine  are  then  added,  and  finally  a  gram  of 
phenol  to  each  lOO  grams  of  the  mixture.  The  whole  is 
then  warmed  for  lo  to  15  minutes  with  constant  stirring, 
until  the  fluid  is  quite  clear,  and  is  finally  filtered  through 
glass  wool  or  filter-paper.  This  may  evidently  be  best  done 
with  the  aid  of  a  hot-water  filtering  apparatus. 

57.  Less  delicate  objects,  like  sections  of  wood  and  the 
like,  may  be  transferred  directly  from  water  to  glycerine- 
gelatine  ;  more  delicate  preparations  should  first  be  brought 
into  glycerine.  This  may  be  accomplished,  in  case  of 
objects  which  collapse  very  easily,  by  placing  them  in  a  \o% 
solution  of  glycerine,  which  is  then  allowed  to  concentrate 
gradually  by  standing  in  the  air. 

58.  Since  the  glycerine-gelatine  (glycerine  jelly)  is  solid  at 
ordinary  temperatures,  it  must  be  warmed  before  use  until 
it  becomes  fluid  ;  and  for  this  purpose  the  parafifine  bath 
may  be  used  (§  47).  Or  one  may  prepare  small  pieces  of  the 
jelly,  each  of  suitable  size  for  one  preparation,  and  melt 
them  upon  the  slides.  Such  pieces  may  be  readily  prepared 
by  allowing  a  quantity  of  the  jelly  to  harden  upon  a  plate 
in  a  thin  layer,  w^hich  is  then  cut  into  blocks. 

59.  If  annoying  air-bubbles  occur  in  the  preparation  en- 
closed in  glycerine-gelatine,  they  can  be  easily  removed  from 
objects  not  too  delicate  by  heating  the  jelly  to  boiling. 

Since  preparations  in  glycerine-gelatine  usually  shrink 
pretty  strongly  when  kept  for  a  long  time,  it  is  generally 
advisable  to  seal  them  with  a  cement  ring  ;  but  it  is  best, 
especially  with  thick  sections,  to  apply  this  ring  after  some 
time,  as  otherwise  the  cover-glass  is  easily  broken  by  the 
subsequent  concentration  of  the  jelly,  and  it  is  easier  to 
remove  by  warming,  any  air-bubbles  that  may  appear.  For 
demonstration  preparations  I  apply  the  cement  only  after  a 
year. 

59a.  Chloral-hydrate  gelatine  is  recommended  by  Geoff  roy 
(I)  as  a  mounting  medium.  It  is  prepared  by  dissolving  3 
to  4  grams  of  good  gelatine  in  100  ccm.  of  a  10^  aqueous 
solution  of  chloral  hydrate,  at  as  low  a  temperature  as  pos- 


GENERAL   METHODS.  43 

sible.  The  sections  are  placed  directly  in  this  fluid,  and, 
since  a  thin  layer  of  gelatine  is  soon  formed  at  the  edge  of 
the  cover-glass  by  the  evaporation  of  water,  the  preparation 
may  be  sealed  after  a  short  time  with  maskenlack  or  an 
alcoholic  solution  of  sealing-wax.  Many  stains,  such  as 
those  with  iodine  green  or  carmine,  are  well  preserved  in 
this  medium. 

60.  Enclosure  in  Air. — Ash  skeletons,  crystals  easily  solu- 
ble in  water,  and  the  like  may  be  often  best  preserved  sim- 
ply dry.  To  protect  them  from  dust  it  is  also  necessary  in 
such  cases  to  cover  the  objects  with  a  cover-glass.  This 
may  be  attached  to  the  slide  by  wax  or  paraffine  around  its 
edges  or  even  with  gummed  paper. 

61.  TJie  observation  of  crystalline  precipitates  and  the 
like  is  best  conducted  in  the  air  by  ordinary  light ;  while  in 
polarized  light  the  interference  colors  appear  most  pure  on 
enclosure  in  a  strongly  refractive  medium  like  Canada  bal- 
sam. Instructive  preparations  of  both  kinds  may  be  pre- 
pared by  placing  on  the  slide  a  drop  of  Canada  balsam  so 
small  that  it  occupies  only  a  part  of  the  space  beneath 
the  cover-glass,  leaving  a  part  of  the  crystals  in  air.  To 
exclude  destructive  agencies  so  far  as  possible,  the  edge  of 
the  cover-glass  may  then  be  surrounded  with  paraffine  or 
wax. 

62.  Sealing  Media. — Of  the  numerous  sealing  media  pro- 
posed by  various  authors  may  be  mentioned  here  first  the 
so-called  "gold-size,"  which  is  well  adapted  for  glycerine 
and  glycerine-gelatine  preparations.  Since  the  method  of." 
preparing  it  is  quite  elaborate,  it  is  best  to  obtain  it  ready- 
prepared  (e.g.,  from  Dr.  G.  Griibler,  Leipzig). 

For  glycerine-gelatine  preparations  Canada  balsam,  asphalt 
varnish,  and  maskenlack  N.  Ill  are  also  well  adapted.  The 
cover-glass  cement  containing  amber,  recommeded  by  Hey- 
denreich,  affords  a  very  trustworthy  medium  ;  but  it  should 
not  be  colored  with  eosin,  as  was  the  case  with  a  prepara- 
tion formerly  furnished  by  Dr.  G.  Griibler,  because  this 
gradually  goes  over  into  the  glycerine-gelatine  and  ma^^r 
cause  an  unpleasant  staining  of  the  preparation. 


IPart  Second 

MICROCHEMISTRY. 

A.  Inorganic  Compounds. 
I.  Oxygen,  Oa. 

63.  For  the  microchemical  recognition  of  oxygen,  the 
method  with  Bacteria  devised  by  Engelmann  (I)  may  often 
be  used  with  success.  This  depends  upon  the  fact  that 
moving  Bacteria  at  once  cease  their  motion  if  oxygen  is 
withdrawn  from  them,  and  immediately  resume  it  on  the 
subsequent  renewal  of  the  oxygen  supply.  Oxygen  also 
affects  the  direction  of  motion  of  Bacteria,  since  they  move 
toward  the  fluid  which  is  richest  in  oxygen. 

It  is  easy  to  satisfy  one's  self  of  this  by  placing  a  drop  of 
fluid  containing  moving  Bacteria  on  a  slide,  and  covering  it 
with  a  large  cover-glass.  The  oxygen  of  the  fluid  is  soon 
exhausted  and  the  motion  continues  only  at  the  edges  of 
the  cover,  or  around  included  air-bubbles,  which  are  espe- 
•cially  instructive.  It  may  also  soon  be  seen  that  the  Bacteria 
group  themselves  in  heaps  at  these  places. 

The  sensitiveness  of  this  reaction,  which  shows  very  small 
quantities  of  oxygen,  is  naturally  dependent  in  some  degree 
upon  the  choice  of  Bacteria.  Those  which  are  obtained  by 
letting  split  peas  decay  in  water  are  very  useful.  After  a 
few  days  innumerable  Bacteria  appear,  which  are  commonly 
called  Bacterinui  tcrvio. 

It  may  be  added,  with  reference  to  the  management  of 
the  reaction,  that  it  is  usually  desirable  to  use  large  cover- 
•^lasses,  whose  edges  may  be  sealed  with  cacao-butter,  wax, 

44 


MICROCHEMISTRY.  45 

or  paraffine  to  prevent  the  evaporation  of  the  fluid  and  the 
access  of  oxygen. 


2.  Peroxide  of  Hydrogen,  HjOa. 

64.  For  testing  living  Spirogyrce  for  the  presence  of  perox- 
ide of  hydrogen,  Bokorny  (III)  used  the  two  following 
methods. 

The  first  is  based  on  the  fact  that  peroxide  of  hydrogen 
in  the  presence  of  iron  sulphate  at  once  sets  iodine  free  from 
potassium  iodide,  so  that  any  starch  or  starch-paste  present 
is  colored  blue.  He  therefore  placed  Spirogyra  cells  con- 
taining starch  in  a  very  dilute  solution  of  ferrous  sulphate 
and  potassium  iodide,  and  deduced  the  absence  of  peroxide 
of  hydrogen  from  the  failure  of  the  starch-grains  to  become 
colored  blue.  This  was  emphasized  by  the  intense  bluing 
of  the  starch  in  threads  which  had  previously  been  saturated 
with  the  peroxide. 

In  the  second  method,  Bokorny  acted  upon  the  fact  that 
tannin  which  gives  a  blue  reaction  with  ferric  salts  is  at 
once  turned  blue  by  ferrous  sulphate  in  the  presence  of 
peroxide  of  hydrogen,  while  the  blue  color  otherwise  appears 
only  after  some  time  in  consequence  of  the  gradual  oxida- 
tion of  the  ferrous  salt  in  the  air.  He  observed,  in  agree- 
ment with  the  above,  that  Spirogyra  threads  containing  a 
tannin  that  reacts  with  ferric  salts  became  blue  only  many 
hours  after  being  placed  in  a  solution  of  ferrous  sulphate, 
while  the  blue  color  appeared  at  once  in  threads  saturated 
with  the  peroxide. 

Pfeffer  has  (IV,  446)  questioned  the  conclusiveness  of 
these  experiments  and  especially  doubted  whether  the  dilute 
reagents  used  by  Bokorny  were  really  taken  up  by  the  living 
cells.  But  Bokorny  has  (I  and  II)  recently  made  observa- 
tions which  show  that  the  ferrous  sulphate  is  really  taken  up 
by  the  living  cells,  and  the  conclusiveness  of  the  second  reac- 
tion cannot,  therefore,  be  doubted. 

65.  Pfeffer  (IV)  was  led  by  more  extended  observations 
to  the  conclusion  that  peroxide  of  hydrogen  does  not  occur 


A 


46  BOTANICAL   MICROTECHNIQUE. 

within  the  living  cell.  He  showed  first  that  the  peroxide 
may  be  taken  up  by  living  cells  without  harm  and  that  it 
often  produces  in  them,  even  when  in  very  small  quantity^ 
plainly  visible  reactions,  which  do  not  otherwise  occur  in 
living  cells. 

Pfeffer  used  first  for  these  researches  plants  whose  color- 
less cell-sap  is  colored  by  the  oxidizing  effect  of  the  peroxide, 
as,  e.g.,  the  epidermal  cells  of  the  stem  and  root  of  seedlings 
of  Vicia  Faba  or  the  root-hairs  of  Trianca  bogotensis.  In 
these  the  peroxide  produces  a  browning  of  the  cell-sap  which 
is  usually  followed  by  the  separation  of  red-brown  or  almost 
black  granular  masses,  as  is  shown  in  Fig.  17. 
Here  is  figured  a  part  of  an  epidermal  cell  from 
the  stem  of  Vicia  Faba^  which  has  lain  five  hours 
in  a  solution  of  peroxide  of  hydrogen,  prepared 
by  mixing  ten  parts  of  pure  water  with  one  part 
of  a  commercial  peroxide  solution  already  six 
months  old. 

66.  Pfeffer  also  worked  with  cells  which  have 
naturally  a  colored  cell-sap,  like  the  stamen-hairs 
of  Tradescantia  virginica.  In  this  case  the  blue 
Fig.  i7.-Partof  ccll-sap  is  wholly  blcachcd  by  the  peroxide  or 
cell  *^of  ^'t'h^  takeSz-a  yellow-brown  or  vinous-yellow  color. 
\aba,  hJe  Blcaching  by  the  peroxide  taken  up  may  also 
being  placed  bc  obscrvcd  in  cclls  whosc  protoplasm  has  been 
hydrogen  so-  previously  colored  blue  by  cyanin.     The   root- 

lution. 

hairs  of  Trianca  bogotensis  are  well  adapted  for 
these  experiments.  In  their  protoplasm,  when  in  a  very 
dilute  solution  of  cyanin,  prepared  by  warming  that  dye 
with  water,  various  blue  differential  stains  were  evident  in 
from  three  to  fifteen  minutes,  and  were  destroyed  by  perox- 
ide of  hydrogen  in  less  than  a  minute. 

67.  It  may  be  remarked  that  Pfeffer  worked  with  solu- 
tions of  from  .015^  to  i^  of  the  peroxide.  Since  the  com- 
mercial peroxide  always  contains  some  free  hydrochloric 
acid  to  increase  its  keeping  quality,  it  must  be  neutralized 
with  sodium  bicarbonate ;  and  Pfeffer  adds  this  in  slight 
excess. 


MICROCHEMISTR  V. 


47 


3.   Sulphur,  S. 

68.  The  sulphur  which  occurs  in  various  Bacteria  in  the 
form  of  strongly  refractive  spheres  (cf.  Fig.  18,  i,  a~c)  is, 
according  to  Cohn  (I,  178),  insoluble  in  water  and  hydro- 
chloric acid,  but  soluble  in  an  excess  of  absolute  alcohol,  in 
hot  potash,  or  in  sodium  sulphite.  Nitric  acid  and  potassium 
chlorate  dissolve  them  at  ordinary  temperatures,  as  does  car- 
bon bisulphide  ;  but  the  entrance  of  the  latter  into  the  cells 
of  the  Bacteria  must  be  aided  by  previously  killing  them 
with  sulphuric  acid  or  by  drying.  According  to  Wino- 
gradsky  (I,  521),  this  solubility  in  carbon  bisulphide  is  not 
complete,  although  the  insoluble  residue  in  this  reagent  is 
always  small.  According  to  Biitschli  (I,  6),  the  granules  of 
sulphur  are  soluble  in  twenty-four  hours  in  artificial  gastric 
juice,  or  in  a  10^  soda  solution. 

69.  Various  observations  of  Winogradsky  (I,  518)  ex- 
plain the  accumulation  of  the  sulphur  granules.  Accord- 
ing to  this  author,  they  are 
always  quite  spherical  in 
the  living  cell  and  run  to- 
gether on  the  death  of  the 
cells,  for  example,  on  heat- 


very  rich 

hours'"  cul- 


ing  to  70°  C,  into  large 
drops  which  change  into 
beautiful  crystals  of  sul- 
phur. This  crystallization 
takes  place  best  when  Beggi- 
atoa  threads  rich  in  sulphur 
are  placed  for  about  a  min- 
ute in  a  concentrated  aque- 
ous  solution   of  picric  acid 

and  then  washed  in  a  large  quantity  of  water.  On  such 
threads  beautifully  formed  sulphur  crystals  were  found  after 
twenty-four  hours,  partly  monoclinic  prisms  and  partly  rhom- 
bic octahedra  (cf.  Fig.  18,  2).  It  is  therefore  to  be  presumed 
that  these  sulphur  grains  consibt  of  the  modified  form  of 
sulphur  which  is  semi-fluid  or  oil-like  at  ordinary  tempera- 


FiG.  18.— I.  Beggiatoa  threads, 
in  sulphur;  b,  after  24,  f,  after 
ture  in  spring-water;  s,  granules  of  sulphur. 

2.  The  same,  24  hours  after  treatment  with 
picric  acid,  which  largely  converts  the  sul- 
phur globules  into  crystals.  After  Wino- 
gradsky. 


48 


BOTANICAL   MICROTECHNIQUE, 


tures.  In  fact,  the  precipitate  of  sulphur  which  is  formed 
when  dilute  hydrochloric  acid  is  added  to  a  solution  of 
calcium  pentasulphide  shows  the  same  relations,  on  micro- 
scopic examination,  as  the  sulphur  granules  of  the  Beggia-- 
toas.  It  is  possible,  according  to  Winogradsky,  that  these,, 
as  well  as  the  granules  of  the  precipitated  sulphur,  gradually- 
pass  over  into  the  solid  condition,  and  that,  especially  in 
slowly  growing  threads,  all  the  stages  from  the  fluid  to  the 
almost  solid  condition  occur. 

70.  It  should  be  observed  here  that  Jonsson  (I)  has  seen 
in  a  mycelium  of  Penicillium,  growing  on  dilute  sulphuric 
acid,  strongly  refractive  bodies  which  correspond  in  many 
of  their  reactions  with  the  sulphur  granules  of  the  Beggia- 
toaSy  and  consist,  according  to  Jonsson,  of  a  mixture  of  suU 
phur  and  an  oil-like  substance. 


4.  Hydrochloric  Acid,  HCl,  and  its  Salts. 

71.  For  the  recognition  of  hydrochloric  acid  Schimper 
(II,  212)  found  the  two  following  methods  especially  useful. 
I.  The  addition  of  silver  nitrate  causes  the  formation  of 
amorphous  silver  chloride,  but  this  may  be  obtained  in  crys- 
talline form  by  dissolving  the  precipitate  arising  from  the 
addition  of  silver  nitrate  in  as  little  ammonia  as  possible  and 
allowing  the  fluid  to  evaporate.  Regular  crystals  of  silver 
chloride  are  thus  formed,  consisting  chiefly  of  hexahedra,^ 
octahedra,  and  rhombic  dodecahedra,  as  well  as  combina- 
tions of  these  (cf.  Fig.  19).  These 
crystals  gradually  become  violet-col- 
ored in  the  light ;  but  in  the  presence 
of  reducing  plant-juices  they  often  be- 
come very  rapidly  colored.  Formed 
silver  chloride  may  also  be  recognized 
Fig.  i9.-crystais  of  silver  bv   its   ready   solubility   in    potassium 

chloride.     After  Haushofer.        ^  ''  '  * 

cyanide,  in  sodium  hyposulphite,  and 
in  a  concentrated  solution  of  mercuric  nitrate.  It  is  also 
somewhat  soluble  in  concentrated  solutions  of  the  alkaline 
metals  and  in  concentrated  hydrochloric  acid ;  and,  accord- 


oo2 


MICROCHEMISTRY.  49 

ing  to  Borodin's  method,*  silver  chloride  may  be  tested  with 
a  concentrated  solution  of  silver  chloride  in  concentrated 
hydrochloric  acid  or  salt  solution  (sodium  chloride), 

II.  Thalliiun  sulphate  causes  at  once,  or  at  least  on  evap- 
oration, the  formation  of  regular  octahedra  or  variously 
shaped  skeletons  of  thallium  chloride,  which  may  be  tested, 
according  to  Borodin's  method,  with  a  concentrated  solu- 
tion of  thallium  chloride. 


5.  Sulphuric  Acid,  H2SO4,  and  its  Salts.  . 

72*  For  the  recognition  of  sulphuric  acid  we  still  lack  a 
completely  trustworthy  method.  The  following  methods 
have  been  used  by  Schimper  (II,  219) : 

1.  Barhun  chloride  always  causes  a  precipitate  of  barium 
sulphate,  but  this  is  rarely  crystalline  and  its  positive  deter- 
mination is  therefore  rarely  possible. 

2.  Strontium  nitrate  causes  the  formation  of  small,  thick 
crystals  of  a  mostly  roundish-rhombic  form,  though  some- 
times sharp  and  with  straight  outlines,  which  are  insoluble 
in  water. 

3.  Potassium  sulphate  often  crystallizes  out  of  a  solution 
of  ash  in  water  in  the  form  of  hexagonal  plates,  which  fall 
into  colorless  granules  on  the  addition  of  barium  chloride. 


*  According  to  Borodin's  method  (II,  805)  a  given  precipitate  soluble  in 
water  is  tested  with  a  completely  saturated  solution  of  the  substance  that  is 
suspected  in  it.  If  the  suspicion  is  correct,  the  precipitate  will  not  be  dis- 
solved, while  any  other  substance,  unless  some  reaction  occurs,  will  be  solu- 
ble. If,  for  instance,  we  have  to  do  with  a  mixture  of  asparagin  and  saltpeter 
(potassium  nitrate)  the  asparagin  crystals  will,  of  course,  be  insoluble  in  a  con- 
centrated solution  of  asparagin,  but  the  saltpeter  crystals  will  be  dissolved. 
On  the  subsequent  addition  of  water,  asparagin  crystals  will  be  dissolved  also. 
So,  as  in.  the  above-mentioned  case,  silver  chloride  will  be  insoluble  in  a  con- 
centrated solution  of  silver  chloride  in  strong  hydrochloric  acid  (or  NaCl), 
while  it  must  dissolve  on  the  addition  of  more  acid  (or  NaCl  solution).  In 
case  of  substances  not  too  easily  soluble,  this  method  renders  good  service  ir» 
microchemistry  ;  but  great  care  must  be  taken  in  each  case  that  the  solution 
employed  is  really  completely  saturated,  and  that  it  does  not  become  capable, 
through  changes  of  temperature,  of  dissolving  more  of  the  substance  concerned. 


-50 


no  TA  NICA  L   MICRO  TECHXIQ  UE 


v>r  into  heaps  of  red   granules  on  the  addition  of  phitinum 
chloride. 

4.  Sodium  and  potassium  sulphates  may  often  be  recog- 
nized in  the  living  tissues  by  means  of  nickel  sulphate. 
With  this  they  form  well  crystallized  double  salts  of  the 
composition  NiSO,  +  NajSO,  +  6H,0  (or  the  correspond- 
ing K  salt) ;  these  occur  mostly  in  the  form  of  the  mono- 
clinic  prism  combined  with  the  basal  plane,  but  are  pretty 
easily  soluble  in  water. 


6.  Nitric  Acid,  HNOa.  Nitrous  Acid,  HNO,,  and  their  Salts. 

73.  Diphenylaminc  was  first  recommended  by  Molisch  (I) 
for  the  recognition  of  the  nitrates,  and  he  used  for  fresh 
rsections  a  solution  of  from  jL^  to  -^^  of  a  gram  of  it  in  10 
ccm.  of  pure  concentrated  sulphuric  acid,  or  for  dried  sec- 
tions a  concentrated  solution  of  it  in  concentrated  sulphuric 
acid.  In  the  presence  of  nitrates  there  occurs  immediately 
after  the  addition  of  this  reagent  a  deep-blue  coloring 
which,  after  a  time,  disappears  or  passes  into  brownish 
yellow. 

This  reaction  occurs  in  the  same  way  in  the  presence  of 
nitrites,  and  it  can  therefore  be  used  for  the  recognition  of 
nitrates  only  when  the  absence  of  nitrous  salts  is  proved. 
But  in  fact  all  investigations  on  the  subject  heretofore 
have  led  to  the  conclusion  that  nitrous  salts  do  not  occur 
Avithin  the  living  plant ;  and  therefore  this  objection  to  the 
applicability  of  diphenylaminc  as  a  reagent  for  nitrates  falls, 
so  far  as  the  microchemical  study  of  the  plant  is  concerned. 

It  should  be  remarked  that  other  compounds  than  nitrates 
and  nitrites  give  the  same  reaction,  as,  for  example,  man- 
cjanese  peroxide,  potassium  chromate  and  chlorate,  hydro- 
<^^en  peroxide,  ferric  oxide  and  its  salts  (cf.  Frank  I  and  II, 
and  Kreusler  I).  But  these  substances  appear  to  be  as 
rare  in  the  plant  as  nitrites  ;  at  all  events,  plants  freed  from 
nitrates  never  give  a  blue  color  with  diphenylamine,  accord- 
ing to  the  confirmatory  researches  of  Frank  and  Schim- 
perfll,  217). 


MICROCHEMISTRY.  5*1 

It  is  more  important  to  note  that  the  reaction  may 
entirely  fail,  even  with  large  quantities  of  nitrates,  in  pres- 
ence of  various  substances,  as,  for  example,  lignified  cell- 
membranes  (cf.  Schimper  II,  217).  It  follows,  therefore, 
that  the  absence  of  nitrates  can  never  be  deduced  from  a 
negative  result  of  this  test. 

74.  Brucin  gives  a  bright  red  or  reddish-yellow  color  with 
nitrates  and  nitrites,  but  this  gradually  disappears.  Molisch 
(VI,  152)  uses  for  microchemical  purposes  a  solution  con- 
taining .2  gram  of  brucin  in  10  ccm.  of  concentrated  sul- 
phuric acid,  but  remarks  that  this  reaction  is  inferior  in 
clearness   to    the    diphenylamine-reaction. 

75.  According  to  Arnaud  and  Fade  (I),  the  alkaloid 
cinchonamin  (CigH^^N^O),  obtained  from  the  bark  of  Remijia 
purdieana,  may  be  used  for  the  microchemical  recognition 
of  nitrates.  Its  nitrate  is  almost  absolutely  insoluble  in 
acidified  v/ater  and  forms  beautiful,  readily  recognizable 
crystals  whose  form  is,  unfortunately,  not  described  by 
these  authors.  They  immerse  fresh  sections  of  the  parts  to 
be  tested  directly  in  a  .4^  solution  of  the  chloride  of  cin- 
chonamin which  is  slightly  acidified  with  hydrochloric  acid. 
The  crystals  of  nitrate  of  cinchonamin  will  then  be  formed 
within  the  cells  containing  nitrates. 

76.  Potassiitin  nitrate  (KNO3)  ^nay  also  be  recognized  by 
covering  the  sections  with  a  cover-glass,  adding  alcohol, 
and  then  allowing  them  to  dry.  The  saltpeter  then  usually 
crystallizes,  chiefly  in  the  form  of  rhombic  plates  (cf.  Fig. 
27,  §  1 30),  which  stand  out  sharply,  especially  in  polarized 
light.  Asparagin  also  forms  similar  crystals,  but  these  may 
be  easily  distinguished  from  saltpeter  crystals  by  measuring 
their  angles  (cf.  §  130).  Besides,  the  latter  are,  of  course, 
easily  soluble  in  a  concentrated  aqueous  solution  of  aspar- 
agin, and  are  not  destroyed  by  heating.  They  can  also  be 
readily  tested  with  a  solution  of  diphenylamine.  Borodin's 
method  (cf.  §  71,  note)  is  inapplicable,  on  the  other  hand, 
on  account  of  the  ready  solubility  of  potassium  nitrate. 


52  BOTANICAL   MICROTECHNIQUE. 


7.  Phosphoric  Acid,  H3PO4 ,  and  its  Salts. 

77.  The  following  reactions  are  adapted  for  the  micro- 
chemical  recognition  of  phosphoric  acid : 

1.  Nitric  acid  and  amtnonitim  molybdate.  This  reagent, 
first  introduced  into  microchemistry  by  Hansen  (I,  96), 
causes  the  formation  of  regular  crystals  which  represent 
chiefly  a  combination  of  the  octahedron  and  the  cube,  and 
are  colored  an  intense  yellow.  There  is  commonly  no 
danger  of  confusing  these  with  the  isomorphic  compounds 
of  arsenic  acid,  so  far  as  the  study  of  vegetable  objects  is 
concerned. 

It  is  convenient  to  use  as  the  reagent  a  solution  which 
contains  12  ccm.  of  officinal  nitric  acid  of  specific  gravity 
1. 1 8,  to  one  gram  of  ammonium  molybdate.  In  the  pres- 
ence of  small  quantities  of  acid  the  precipitate  is  formed 
only  after  slight  warming  (to  40°-50°  C),  and  then  oftea 
only  after  some  time. 

The  sections  to  be  tested  are  best  burned  before  the 
addition  of  the  reagent,  since  otherwise  the  reaction  may 
be  hindered  by  the  presence  of  certain  organic  substances, 
as,  for  example,  potassium  tartrate.  Besides,  the  phosphoric 
acids  combined  with  the  nuclein  or  otherwise  organically 
united,  as,  for  instance,  the  phosphoric  acids  contained  in 
the  globoids,  are  not  directly  shown  by  this  reagent,  but 
only  in  the  ash  (cf.  Schimper  II,  215).  This  reagent  may 
be  applied  directly  to  the  ash  prepared  by  heating  upon  the 
cover-glass.  Thus  is  obtained  at  once  with  the  ashes  of 
sections  of  not  too  young  stems  of  Stapclia  picta  a  strong 
reaction,  which  occurs  only  after  some  hours  in  sections 
prepared  from  alcoholic  material  which  contain  spha^rites 
of  calcium  phosphate  (cf.  ^  96). 

2.  The  addition  of  magnesium  sulphate  and  ammonium 
chloride  produces  with  salts  of  phosphoric  acid  a  crystalline 
precipitate  of  ammonio-magnesium  phosphate,  w^hich  is 
practically  insoluble  in  ammonia  and  ammonium  chloride 
solutions.     These  crystals,  some  of  the  most  characteristic 


MICROCHEMIS  TRY.  55 

of  which  are  illustrated  after  Haushofer  (I,  92)  in  Fig.  20^ 
belong  to  the  rhombic  system.  A  similar  salt  is  also  formed 
by  arsenic  acid. 

A  suitable  reagent  may  be  obtained  by  mixing  25  vol- 
umes of  a  concentrated  aqueous 
solution  of  magnesium  sulphate,  2 
volumes  of  concentrated  aqueous 
solution  of  ammonium  chloride,  and 
15  volumes  of  water.  If  there  be 
placed  in  this  solution  sections  from 
alcoholic  material  of  the  stem  of 
Stapelia  picta,  which  have  previously  F'g.  20.  —  crystals  of  ammonio- 

J:  ^  '  A  •        magnesium  phosphate.     After 

been  soaked  for  a  time  in  water  to  Haushofer. 
prevent  the  formation  of  a  precipitate  by  the  alcohol,  there 
appear  after  a  time  in  the  immediate  vicinity  of  the  sphae- 
rites  of  calcium  phosphate,  in  consequence  of  their  gradual 
solution,  well  formed  crystals  of  ammonio-magnesium  phos- 
phate, among  which  the  X-shaped  skeleton-crystals  appear 
to  be  especially  characteristic.  This  reaction  may  be 
hastened  by  warming,  but  the  crystals  are  then  less  regu- 
larly formed. 

For  the  recognition  of  phosphoric  acid  within  the  tissues^ 
this  reaction  is,  according  to  Schimper  (II,  216),  preferable 
to  the  previously  described  one,  since  it  is  not  interfered 
with  by  the  presence  of  organic  compounds  and  is  very 
delicate. 

8.  Silicic  Acid,  SiO^,  and  the  Silicates. 

78.  Silicic  acid  occurs  in  the  vegetable  kingdom  partly  in 
incrustations  of  cell-membranes  and  partly  in  the  form  of 
variously-shaped  silica  masses  in  the  interiors  of  cells  (cf. 
Kohl's  compilation,  II,  197). 

For  the  microchemical  recognition  of  silicic  acid,  one  may 
utilize  its  peculiarity  of  not  being  changed  by  heating.  Its 
insolubility  in  all  acids  except  hydrofluoric  acid  serves  to 
distinguish  it  from  other  inorganic  substances.  In  case  of 
some  strongly  silicified  organs  it  is  possible  by  the  combined 


54  BOTANICAL   MICROTECHNIQUE. 

action  of  acids  and  heat  to  obtain  completely  coherent 
siliceous  membranes,  the  so-called  silica  skeletons.  From 
the  membranes  of  the  diatoms,  which  are  peculiarly  rich  in 
silicic  acid,  or  from  the  epidermis  of  the  Graminece  or  Eqtiu 
jetacece*j  beautiful  siliceous  skeletons  maybe  obtained  by 
treating  them  as  proposed  by  Sachs.  This  method  consists 
in  heating  the  organ  or  organism  on  a  cover-glass,  or  on  a 
bit  of  mica  to  prevent  the  residue  from  adhering,  with  a 
drop  of  concentrated  sulphuric  acid  until  the  ash  remaining 
after  the  evaporation  of  the  acid  has  become  quite  white. 

In  case  of  objects  poorer  in  silicic  acid,  satisfactory 
siliceous  skeletons  cannot  usually  be  obtained  by  this  simple 
method.  It  is  then  commonly  better  to  remove  the  soluble 
inorganic  substances  from  the  pieces  before  burning  by 
treatment  with  hydrochloric  or  nitric  acid.  In  this  way 
pure  white  skeletons  may  be  much  more  easily  obtained 
and  may  be  freed  from  foreign  admixtures  by  renewed 
treatment  with  hydrochloric  acid. 

79.  Besides,  siliceous  skeletons  may  be  very  well  prepared 
wholly  in  the  wet  way  by  the  method  proposed  by  Mil- 
iarakis  (I).  The  object  is  first  treated  in  a  beaker  with  con- 
centrated sulphuric  acid  until  it  is  quite  black  and  then  a 
20%  aqueous  solution  of  chromic  acid  is  added.  In  this 
mixture  suberized  membranes  are  also  wholly  destroyed, 
and  only  the  siliceous  skeletons  remain  behind.  They  may 
then  be  easily  isolated,  after  the  addition  of  water,  by  de- 
canting, and  may  be  completely  cleaned  by  repeated  wash- 
ing with  water  and  alcohol.  The  siliceous  skeletons  of 
<liatoms  obtained  in  this  way  show,  especially  when  exam- 
ined in  air,  the  finest  structural  features  of  their  membranes. 

According  to  Kohl  (II,  226)  this  method  is  applicable 
only  where  considerable  quantities  of  silicic  acid  are  present. 
This  author  obtains  very  delicate  siliceous  skeletons  by 
burning  from  parts  of  plants  with  a  small  proportion  of 
•silicic  acid,  which  would  be  completely  dissolved  by  the 
treatment  with  chromic  sulphuric  acid.     In  other  cases  the 


*[Our  Equisetum  hiemale  is  especially  good  for  this  purpose.] 


MICROCHEMIS TR  Y.  55 

presence  of  silicic  acid  can  only  be  recognized  in  the  ash  hy 
the  sodium  silico-fluoride  reaction  (cf.  §  81). 

80.  To  test  the  skeletons  obtained  by  either  of  these 
methods  for  the  presence  of  silicic  acid,  hydrofluoric  acid 
may  be  used,  in  which  pure  silica-skeletons  should  dissolve, 
completely. 

Karner  (I,  262)  recommends  for  this  purpose  a  dilute 
aqueous  solution  of  hydrofluoric  acid,  which,  as  it  attacks, 
glass,  must  be  kept  in  a  bottle  of  rubber  or  lead,  and  must 
be  placed  upon  the  objects  to  be  tested  with  a  platinum 
wire  or  a  rubber  rod.  The  sHde  which  supports  the  object 
must,  of  course,  be  protected  from  the  action  of  the  acid,, 
and  for  this  purpose  covering  with  Canada  balsam,  glue, 
vaseline,  or  glycerine  has  been  recommended.  But,  accord- 
ing to  Karner  (I,  262),  it  is  most  convenient  to  cover  the 
slide  with  a  piece  of  transparent  sheet-wax,  which  must  be 
first  somewhat  warmed  and  smoothed  by  rubbing  between 
the  hands.  Instead  of  a  cover-glass  this  author  recommends 
tha  use  of  gelatine  paper.  He  also  fastens  a  bit  of  the  same 
paper  to  the  objective  with  Canada  balsam,  to  protect  it 
from  the  vapor  of  hydrofluoric  acid. 

When  Karner  (I,  266)  allowed  hydrofluoric  acid  to  act 
upon  membranes  not  previously  treated  with  some  acid  01 
the  like,  he  usually  found  only  a  partial  solution  of  the 
silicic  acid.  Whether  this  was  due  to  the  physical  action  of 
other  constituents  of  the  membrane  or  to  a  chemical  union> 
perhaps  of  silicium  with  cellulose,  is  not  yet  certain. 

81.  Besides  its  solubility  in  hydrofluoric  acid,  one  may 
use  for  the  recognition  of  silicic  acid  the  formation  of  crys- 
tals of  sodium  silico-fluoride,  which  are  with  great  difficulty 
soluble  in  water.  To  obtain  these  crystals  hydrofluoric  acid 
and  some  sodium  chloride  are  added  to  the  ash  and  allowed 
to  slowly  evaporate.  The  crystals  of  sodium  silico-fluoride 
which  then  form  if  siHcium  is  present  belong  to  the  hex- 
agonal system  and  represent  chiefly  combinations  of  prisms 
and  pyramids,  or  of  these  with  six-sided  plates  also.  In 
stronger  solutions  six-rayed  stars  and  rosettes  are  also  ob- 
served as  skeleton  forms  (Haushofer,  I,  98). 


56  BOTANICAL   MICROTECHNIQUE. 

9.  Potassium,  K. 

82.  Since  ammonium  cannot  occur  in  the  ^s\\,  platinum 
chloride  may  well  serve  for  the  recognition  of  potassium. 
The  potassium-platinum  chloride  thus  formed  crystallizes  in 
regular  octahedra  and  cubes.  According  to  Schimper  (II, 
213)  the  ash  is  dissolved  in  a  drop  of  acidified  water,  is 
warmed  until  dry,  and  the  reagent  is  added  before  or  after 
cooling.  But  the  reagent  used  must  first  be  tested  with 
much  care  to  show  that  it  is  really  free  from  potassium. 
This  may  be  done  by  letting  a  drop  of  the  reagent  slowly 
evaporate  on  the  slide. 

10.  Sodium,  Na. 

83.  The  nranyl-magnesiiim  acetate  recommended  by 
Streng  (I)  serves  excellently  for  the  recognition  of  very 
small  quantities  of  sodium.  It  forms  with  sodium  a  double 
salt  of  the  composition  CH,CO,Na +  (CH,CO,XUO,+ 
{CH,COO),Mg  +  (CH,COO),UO  +  9H,0.  This  compound, 
very  poor  in  sodium  and  therefore  formed  in  the  presence 
of  very  small  quantities  of  sodium,  forms  small  colorless  or 
very  pale  yellowish  rhombohedral  crystals,  which  are  little 
soluble  in  water  and  almost  insoluble  in  alcohol. 

Since  the  solution  of  the  uranium  salt  extracts  sodium 
from  glass  vessels  on  long  standing,  Streng  (III)  recom- 
mends the  direct  addition  of  the  solid  magnesium-uranyl 
salt. . 

Schimper  (II,  215)  used  tiranyl  acetate  for  the  recogni- 
tion of  sodium,  as  it  causes  the  formation,  on  evaporating, 
of  sharply  developed  tetrahedra  of  sodium-uranyl  acetate 
(CH,COONa+(CH3COOXUO),  of  which  the  larger  ones 
appear  faintly  yellowish.  In  the  presence  of  very  small 
quantities  of  sodium  simultaneously  with  magnesium  there 
is  formed,  of  course,  the  above  mentioned  uranyl-magnesium- 
sodium  acetate. 


MICROCHEMIS TR  V.  $7 


II.  Ammonium,  NH*. 


84.  The  so-called  Nesslers  reagent  may  be  used  for  the 
recognition  of  ammonium,  according  to  Strasburger  (I,  74). 
It  is  prepared  in  the  following  manner :  2  grams  of  potas- 
sium iodide  are  dissolved  in  5  ccm.  of  water,  and  then 
mercuric  iodide  is  added  to  the  solution  while  warm,  until 
a  part  remains  undissolved.  After  the  fluid  is  cooled  it  is 
diluted  with  20  ccm.  of  water,  allowed  to  stand  for  a  time, 
filtered,  and  20  ccm.  of  the  filtrate  are  diluted  with  30  ccm. 
of  a  concentrated  caustic  potash  solution.  If  the  fluid  then 
becomes  turbid,  it  must  be  filtered  again  (Nickel  I,  94). 

In  the  presence  of  ammonium  this  solution  takes  a  yel- 
low color,  and  with  more  ammonia  a  brown  precipitate  is 
formed.  But  various  organic  compounds  give  the  same 
reaction  (Nickel  I,  94). 

12.  Calcium,  Ca. 

85.  Calcium  occurs  very  often  within  the  living  plant  in 
crystalline  form,  and  these  crystals,  which  are  met  with 
sometimes  in  the  cell-sap,  sometimes  within  the  membrane, 
consist  most  commonly  of  calcium  oxalate ;  crystals  of 
calcium  carbonate,  gypsum,  and  calcium  tartrate  are  less 
often  observed.  Besides,  calcium  carbonate  often  incrusts 
cell-membranes  in  greater  quantities  ;  and  finally,  calcium 
phosphate  has  been  recognized  in  the  vegetable  organism. 
We  will  describe  first  the  methods  for  recognizing  the 
various  calcium  salts,  and  then  the  methods  of  recognizing 
the  presence  of  calcium  in  the  ash  and  in  the  cell-sap. 

a.  Calcium  Oxalate,  Ca(COO)a. 

86.  Nearly  all  crystals  which  occur  within  the  plant-cell 
consist  of  calcium  oxalate.  They  are  found  partly  in  the 
cell  contents,  and  are  partly  within  or  upon  the  wall.  They 
belong  partly  to  the  tetragonal,  partly  to  the  monoclinic 
crystal  system.     Their  most  important  forms  are  illustrated 


5« 


BOTANICAL   MICROTECHXIQUE. 


in  Kig.  21.  Here  Fig.  I  shows  a  tetragonal  pyraniid,  Figs. 
II  and  III  combinations  of  pyramid  and  prism,  Fig.  IV 
a  monosymmetric  rhombohedron,  Fig.  V  a  rhombic  plate. 
Fig  VI  probably  a  combination  of  positive  and  negative 
hemipyramidswith  the  basal  plane,  Fig.  VII  a  combination 
of  the  rhombic  plate  (Fig.  V)  with  the  clinopinacoid,  Fig. 


H  ^1  ^ 


Fig.  21.— Crystals  of  calcium  oxalate.  I-III.  from  the  spongy  parenchyma  of  Tra^ 
dttcantia  discolor ;  IV,  from  Cycas  circinalis ;  V.  Alusa  paradisiaca  :  VI,  Citrus  vul- 
garis: VII  and  IX,  Cuaiacum  ojfficinale :  VIII,  Citrus  medica.  IV,  V,  VII,  IX  after 
Holzoer  ;  VI  after  Plitzner. 

VIII  a  combination  of  the  rhombohedron  (Fig.  IV)  with 
a  hemipyramid,  Fig.  IX  a  twin  crystal  whose  angle  xyz 
measures  141°  3',  according  to  Holzner  (I,  34).  Calcium 
oxalate  is  also  especially  common  in  the  form  of  fine 
needles  ("  raphides ")  or  tiny  slivers  ("  crystal  sand  ")  on 
which  no  crystallographically  determinable  faces  or  angles 
can  be  recognized ;  and  spha^rocrystals  have  been  seen 
(cf.  Kohl's  compilation,  II,  15). 

Calcium  oxalate  is  insoluble  in  water  and  acetic  acid  ;  but 
in  hydrochloric  acid  it  is  soluble,  though  the  solution  of  the 
larger  crystals,  especially  if  they  are  imbedded  in  mucilage, 
does  not  occur  at  once.     It  is  best  to  place  the  preparations 


MICROCHEMISTRY.  59 

in  concentrated  hydrochloric  acid  and  to  follow  the  solution 
with  a  polarizing  microscope.  The  action  of  the  acid  can 
be  very  much  hastened  by  warming. 

With  nitric  acid  calcium  oxalate  behaves  essentially  as 
with  hydrochloric  acid.  It  is  readily  soluble  in  the  former, 
especially  on  warming. 

87.  By  sulphuric  acid  calcium  oxalate  is  changed  into 
calcium  sulphate  (gypsum),  which  is  little  soluble  in  water 
or  sulphuric  acid,  and  separates  chiefly  in  the  form  of 
needles.  An  immediate  transformation  of  the  calcium 
oxalate  into  gypsum  occurs  if  the  sections  containing  it  are 
placed  directly  in  concentrated  sulphuric  acid  or  in  a  mix- 
ture of  equal  parts  of  water  and  concentrated  sulphuric 
acid,  and  heated  nearly  or  quite  to  boiling.  The  gypsum 
is  then  formed  within  the  same  cells  which  formerly  con- 
tained the  calcium  oxalate  crystals ;  and  each  more  or  less, 
opaque  mass  of  sometimes  plainly  needle-shaped,  sometimes 
more  granular,  particles  of  gypsum  usually  possesses  exactly 
the  same  form  as  the  original  crystal.  These  crystalline 
conglomerates  glisten  brightly  under  the  polarizing  micro- 
scope. 

For  distinguishing  calcium  oxalate  from  calcium  sulphate,. 
Kohl  has  recently  (II,  194)  proposed  a  solution  of  barium 
chloride,  which  leaves  the  oxalate  unchanged,  while  gypsunV 
crystals  become  covered  by  a  finely  granular  layer  of  barium 
sulphate.  In  a  mixture  of  barium  chloride  and  hydrochloric 
acid,  gypsum  is  rapidly  converted  into  barium  sulphate,, 
while  calcium  oxalate  crystals  disappear  in  the  same  mix. 
ture  without  forming  any  precipitate. 

On  treatment  with  caustic  potash  solution,  calcium  oxalate 
at  first  remains  unchanged;  but,  as  Sanio  (I,  254)  first 
observed,  its  crystals  are  suddenly  dissolved  after  some  time, 
usually  after  several  hours,  and  new  crystals  are  formed  in 
the  fluid,  which  have  the  form  of  six-sided  plates  whose 
chemical  composition  is  not  yet  determined. 

88.  On  burning  calcium  oxalate  crystals,  which  can  oest 
be  done  on  a  cover-glass  laid  on  platinum  foil,  the  oxalate 
is  changed  first  into  calcium  carbonate   and  then  into  caU 


6o  BOTANICAL  MICROTECHNIQUE. 

cium  oxide.  The  crystals  preserve  their  original  form,  but 
become  opaque  and  therefore  appear  black  by  transmitted 
light,  but  pure  white  by  reflected  light  (or  dark-field  illumi- 
nation). If  the  crystals  dissolve,  after  the  burning,  in  dilute 
acetic  acid  or  concentrated  hydrochloric  acid,  without  the 
formation  of  gas-bubbles,  this  shows  that  an  oxalate  has 
been  changed  to  the  oxide  ;  while  the  carbonate  dissolves  in 
hydrochloric  acid  with  the  liberation  of  carbonic  acid. 

89.  The  finding  of  calcium  oxalate  crystals  can  be  made 
much  easier  by  examination  by  polarized  light.  They  are 
distinguished  in  general  by  their  strong  double  refraction, 
which  is,  however,  much  greater  in  those  of  the  monoclinic 
system  than  in  those  of  the  tetragonal  system.  The  latter, 
naturally,  cannot  glisten  in  the  polarizing  microscope  with 
crossed  nicols,  when  their  optical  axes  stand  vertical. 

To  make  visible  the  crystals  of  calcium  oxalate  within 
large  organs,  for  example  whole  leaves,  without  cutting 
them  into  sections,  these  may  be  made  quite  transparent. 
For  this  purpose  chloral  hydrate,  which  does  not  attack 
calcium  oxalate,  has  been  used  ;  and  phenol  can  also  be  em- 
ployed. If  the  pieces  are  heated  to  boiling  in  one  of  these 
fluids,  they  usually  become  wholly  cleared  in  a  short  time. 
The  alcoholic  solution  of  sulphurous  acid  used  by  Wehmer 
(I,  218)  for  decolorizing  leaves  will  certainly  be  of  much 
service  in  many  cases. 

For  \\i^  preservation  of  such  preparations  Canada  balsam 
is  best  adapted.  They  may  be  transferred  directly  from 
phenol  to  xylol  and  xylol-Canada  balsam.  The  study  of 
these  cleared  preparations  is  best  conducted  by  polarized 
light. 

b.  Calcium  Carbonate,  CaCO,. 

90.  Calcium  carbonate  rarely  occurs  in  the  interior  of 
cells,  but  is  usually  deposited  in  or  upon  the  cell-wall  (cf. 
Zimmermann  I,  104). 

For  the  recognition  of  the  carbonic  acid  in  calcium  car- 
bonate, acetic  or  hydrochloric  acid  may  be  used.  After  the 
addition  of  one  of  these,  the  carbonic  acid   is  set  free  in 


MICROCHEMIS  TRY.  6 1 

l>ubbles,  as  can  be  directly  observed  under  the  microscope. 
It  has  been  pointed  out  by  Melnikoff  (I,  30)  that  a  concen- 
trated acid*  should  be  used  for  the  recognition  of  small 
quantities  of  carbonic  acid,  and  that  care  should  be  taken 
that  it  reaches  the  bodies  to  be  tested  as  quickly  as  possible. 
Evidently,  the  more  slowly  the  evolution  of  carbonic  acid 
occurs,  the  more  readily  will  it  be  absorbed  by  the  surround- 
ing water  and  carried  away  by  diffusion  without  being  given 
off  in  bubbles. 

91.  For  the  recognition  of  calcium  a  solution  of  animo- 
niiim  oxalate  acidified  with  acetic  acid  may  be  used. 

The  manner  in  which  this  solution  reacts  with  calcium 
salts  is  largely  dependent  upon  its  strength.  For  example, 
I  obtained,  in  sections  of  the  leaf  of  Fiats  elastica,  abundant 
masses  of  crystals  grown  together  in  gland-like  masses  within 
and  near  the  cystolith  cells,  by  placing  them  in  a  solution 
containing  .5^  of  ammonium  oxalate  and  i^fc  of  acetic  acid. 
This  reaction  took  place  at  once  on  placing  the  sections  in 
the  solution,  which  had  previously  been  heated  to  boiling. 
The  crystals  thus  formed  are  strongly  doubly  refractive. 

But  when  a  solution  containing  10^  of  ammonium  oxalate 
and  \io  of  acetic  acid  was  used,  the  oxalate  was  precipitated 
<iirectly  in  the  cystoliths,  which  appeared  quite  unchanged 
on  microscopic  examination.  It  was  only  when  the  sections 
were  placed  in  the  boiling  solution  that  the  cystoliths 
showed  a  more  or  less  granular  structure  on  their  surfaces. 
The  presence  of  a  crust  of  calcium  oxalate  on  the  cystoliths 
<:an  be  easily  shown  by  placing  the  sections  in  pure  10^ 
acetic  acid,  after  washing  out  the  ammonium  oxalate.  The 
still  unchanged  calcium  carbonate  incrusting  the  nucleus  of 
the  cystolith  is  then  dissolved  with  the  formation  of  abun- 
•dant  bubbles  of  gas,  while  the  crust  of  calcium  oxalate  re- 
mains undissolved.  By  the  subsequent  addition  of  hydro- 
chloric acid  the  latter  is  also  dissolved,  so  that  the  pure 
cellulose  skeleton  of  the  cystolith  alone  remains. 


*  Concentrated  HCl  is  best.     Concentrated  acetic  acid  cannot  be  used, 
since  it  dissolves  calcium  carbonate  more  slowly  than  dilute  acid. 


62  BOTANICAL   MICROTECHNIQUE. 

An  aqueous  li  solution  of  oxalic  acid  reacts  in  the  same 
way  as  a  concentrated  solution  of  ammonium  oxalate. 

92.  Calcium  carbonate,  as  well  as  calcium  oxalate,  is 
changed  by  sulphuric  acid  into  gypsum.  It  is  best  in  this 
case  to  use  a  pretty  strongly  dilute  acid.  For  instance,  if 
sections  of  the  leaf  of  Ficus  clastica  are  placed  in  i^  acid,, 
large  masses  of  gypsum  needles  are  formed  in  the  immediate 
vicinity  of  the  cystoliths,  formerly  incrusted  with  calcium 
carbonate. 

Calcium  carbonate  is  not  changed  at  first  by  burning,  but 
is  finally  transformed  into  calcium  oxide. 

c.  Calcium  Sulphate,  CaS04. 

93.  Calcium  sulphate  has  been  recognized  in  many  Des- 
mids  by  A.  Fischer  (I),  and  occurs  in  them  chiefly  in  the 
form  of  tiny  prisms  and  plates,  which  are  sometimes  en- 
closed in  sharply-defined  vacuoles,  as,  for  instance,  in  the 
ends  of  the  cells  of  Closterium  sp.  (cf.  Fig.  22,  a),  or  are  dis- 
tributed throughout  those  parts  of  the  cell  which  contain 
cell-sap  (cf.  Fig.  22,  b).  For  the  microscopical  recognition 
of  gypsum,  A.  Fischer  uses  the  following  reactions  : 

Concentrated  sulpJturic  acid  leaves  gypsum  unchanged  and 
undissolved  when  cold  ;  barium  chloride  transforms  it  into 
barium  sulphate,  which  is  insoluble  in  hydrochloric  and  nitric 


Fig.  m.— «,  the  end  of  a  cf  II  of  Closterium  lunula,  with  jfypsum  crystals  in  the  apical 
vacuole*:  h,  median  lobe  of  Micrasterias  rotata.  The  gypsum  crystals  are  all  col- 
ored black  (x  675).     After  A.  Fischer. 

acid  ;  burning  leaves  the  gypsum  crystals  unchanged.  They 
are  also  insoluble  in  acetic  acid,  but  dissolve  slowly  in  cold 
caustic-potash  solution,  hydrochloric  or  nitric  acid,  or  at 
once  on  heating. 

94.  Hansen  (I,  lo)  has  observed  in  the  leaves  of  various 


MICROCHEMISTR  V.  63 

Marattiacecs  hexagonal  plates,  which,  according  to  the  reac- 
tions carried  out  by  him,  must  consist  of  gypsum  with  an 
admixture  of  magnesium  sulphate.  The  correctness  of  these 
views  has,  however,  been  disputed  by  Monteverde  (II), 
according  to  whom  the  crystals  in  question  consist  merely 
of  calcium  oxalate.  Gypsum  occurs  abundantly,  however, 
according  to  Monteverde,  dissolved  in  the  cell-sap,  and  is 
deposited  in  the  form  of  sphaerocrystals  after  lying  for 
months  in  alcohol.  In  the  same  way,  a  deposit  of  sphaero- 
crystals consisting  of  gypsum  is  produced  in  Hebeclinium 
macrophyllum  by  alcohol,  especially  in  the  young  wood-cells, 
according  to  Hansen  (I,  118). 

d.   Calcium  Tartrate,  CaC2H2.(OH)a.(COO)a. 

95.  In  the  yellowed  leaves  and  petioles  of  Vitis  and 
Ampelopsis,  Schimper  (II,  238)  found  rhombic  crystals  of 
calcium  tartrate,  which  sometimes  reach  considerable  size, 
especially  in  the  parenchyma  of  the  bark  and  pith  of  the 
petiole  of  Vitis  Labrusca,  They  represent  largely  a  combi- 
nation of  the  prism  and  dome,  but  the  most  various  fusions 
also  occur  (cf.  Fig.  23).  These  crystals 
are  very  slightly  soluble  in  water,  but  fH  <]  ^  c:f%> 
very  easily  soluble  in  caustic  potash  solu-  I  i  c^  SX^A) 
tion,  almost  instantly  so  in  a  10^  solution,      |a  "^  *^ 

which  does  not  attack  calcium  oxalate,  p^^,  23._caicium  tar- 
Their  behavior  with  acetic  acid  is  also  ISl^SfXy^^S'l 
characteristic.  Calcium  tartrate  crystals  ?J«?J^,  glth^/ed  Oc" 
are  easily  soluble  in  dilute  solutions  con-  ''^' 
taining  about  2^  of  glacial  acetic  acid,  while  in  the  pure 
glacial  acid,  or  even  in  a  50^  solution  of  it,  they  are  insoluble. 
In  consequence  of  this,  it  may  be  observed,  in  sections  to 
which  concentrated  acetic  acid  has  been  very  gradually 
applied,  that  a  recrystallization  of  previously  dissolved  crys- 
tals occurs. 

Calcium  tartrate  crystals  are  doubly  refractive,  but  this 
power  seems  to  me  much  less  than  that  of  the  monoclinic 
crystals  of  calcium  oxalate.    On  burning  they  are  converted 


64  BOTANICAL   MICROTECHNIQUE, 

into  globular  masses  which  dissolve  in  lo^  acetic  acid  with 
the  formation  of  bubbles. 

e.  Calcium  Malate,  Ca(COO),.CH,.CHOH. 

95a.  Calcium  malate  is  thrown  down  in  large  quantity  hy 
alcohol  in  the  stipes  of  the  fronds  of  Angiopteris  evectUy 
according  to  Belzung  and  Poirault  (I).  It  often  forms 
prisms  of  considerable  size,  which  belong  to  the  rhombic 
system,  and  are  with  difficulty  soluble  in  water,  but  readily 
so  in  acids.  With  sulphuric  acid  they  form  needles  of 
gypsum.  On  heating  on  platinum  foil  they  are  first  black- 
ened, then  show  a  striking  increase  in  volume,  and  are 
finally  converted  into  pure  white  lime.  On  being  heated 
in  the  reducing  flame  they  give  off  the  characteristic  odor 
of  succinic  acid.  By  the  aid  of  Borodin's  method  it  may  be 
shown  that  they  are  completely  insoluble  in  a  saturated  solu- 
tion of  neutral  calcium  malate. 

f.  Calcium  Phosphate,   (CaOjijiPO),  ? 

96.  Calcium  phosphate  has  been  observed  only  in  solu- 
tion in  the  cell,  except  in  case  of  globoids  (cf.  §  388)  and  of 
a  single  instance  which  requires  confirmation  (cf.  Nobbe 
Hanlein  and  Councler  I),  It  separates  in  the  form  of  beau- 
tifully formed  sphaerocrystals  in  the  interior  of  many  parts 
of  plants,  after  they  have  been  placed  in  absolute  alcohol ; 
for  instance,  in  the  stems  of  Euphorbia  caput-meduscB  and 
Stapelia  picta,  as  well  as  in  the  stalk  of  the  frOnd  of  Angi- 
opteris cvccta.  These  sphaerites  are  usually  formed  only 
after  a  considerable  time  (weeks  or  months).^ 

They  have  usually  a  yellowish  or  brownish  color  and  are 
very  slowly  soluble  in  cold  water.  In  hot  water,  too,  they 
are  only  dissolved  after  a  long  time  ;  at  least,  the  solution  of 
large  sphaerites  was  not  complete  after  several  minutes,  when 
they  had  been  heated  to  boiling  in  water  on  the  slide. 

With  ammonia  they  behave  as  with  water ;  they  are  only 

•  I  have  lately  found  globular  or  clustered  bodies  consisting  at  least 
chiefly  of  calcium  phosphate  in  the  living  epidermal  cells  of  a  species  of 
CyperusM.  Zimmermann  VI,  311). 


MICROCHEMIS  TRY.  65 

slowly  soluble  in  acetic  acid,  but  readily  so  in  nitric  and 
hydrochloric  acids,  of  course  without  any  evolution  of  gas. 

In  sulphuric  acid  they  are  quickly  dissolved  with  the  for- 
mation of  gypsum  needles.  If  sections  are  quickly  heated 
on  the  slide  in  a  mixture  of  two  parts  concentrated  sul- 
phuric acid  to  one  part  water,  the  masses  of  gypsum  needles 
then  formed  show  the  same  outline  as  the  sphaerocrystals 
previously  present.  They  may  be  distinguished  from  the 
latter  by  being  wholly  opaque  and  therefore  black  by  trans- 
mitted light,  and  white  by  reflected  light.  But  if  the  sec- 
tions are  placed  in  dilute,  e.g.,  i^,  sulphuric  acid,  the  gyp- 
sum needles  are  gradually  formed  in  the  vicinity  of  the 
sphaerites. 

On  burning,  the  calcium  phosphate  sphaerites  at  first 
become  black  in  consequence  of  the  organic  admixture  to 
be  mentioned  in  the  next  section,  but  on  further  heating 
they  yield  a  pure  white  ash. 

With  nitric  acid  and  ammonium  molybdate,  as  well  as 
with  magnesium  sulphate  and  ammonium  chloride,  they 
give  the  reactions  for  phosphoric  acid  (cf.  §  77). 

When  examined  with  a  polarizing  microscope  these 
sphaerocrystals  show  the  well-known  dark  cross,  with  crossed 
nicols.  By  the  interposition  of  a  gypsum  plate  it  can  be 
determined  that  the  orientation  of  the  optical  axis  is  the 
same  in  them  as  in  starch-grains  and  in  the  sphaerocrystals 
of  inulin. 

In  Canada  balsam  these  sphaerites  may  be  preserved  for 
as  long  as  one  wishes,  and  in  glycerine  gelatine  at  least  for 
a  considerable  time. 

97.  The  various  sphaerocrystals  do  not  represent  an  even 
approximately  chemically  pure  compound,  but  always  con- 
tain a  considerable  quantity  of  organic  substance,  which 
often  forms  an  amorphous  nucleus  at  the  centre  of  each, 
but  is  also  often  contained  in  the  separate  layers.  It  is  to 
be  ascribed  to  this  circumstance  that  calcium  phosphate 
sphaerites  take  up  pretty  freely  various  coloring  matters 
like  methylene  blue  and  borax-carmine  (cf.  Leitgeb  III). 
The  chemical  composition  of  these  organic  substances  is 


66 


BO  TA  XICA  L   MICRO  TECIJNIQ  UE. 


fstill  as  uncertain  as  the  molecular  formula  of  the  calcium 
phosphate  contained  in  the  sphaerocrystals. 


g.  Recognition  of  Calcium  in  the  Ash. 

98.  For  this  purpose  Schimper  (II,  211)  recommends 
especially  the  sulphuric  acid  reaction,  which  may  be  con- 
ducted by  dissolving  the  ash  directly  in  about  2^  sulphuric 
acid  and  then  letting  it  dry  slowly.  There  are  thus  pro- 
duced, especially  at  the  edge  of  the  drop,  crystals  of  gyp- 
sum which  belong  to  the  mono- 
clinic  system.  Among  these, 
plate-like  crystals,  whose  obtuse 
angle  (^,  Fig.  24)  measures  127° 
31',  according  to  Haushofer(I,  33), 
are  especially  characteristic.  Be- 
sides, twin  crystals  are  very  nu- 
merous, whose  edges  form  an  angle 
of  104°  or  of  130°  with  each  other 
(cf.  Fig.  24).  But  the  most  various 
fusions  are  also  found,  on  whose 
projecting  ends  pretty  accurate 
determinations  of  the  angles  may  be  made. 

The  crystals  of  gypsum  are  also  distinguished  by  the  fact 
that  they  are  transformed  instantly  into  small  needles  on 
lieating  in  concentrated  sulphuric  acid.  The  masses  of 
needles  preserve  the  form  of  the  original  crystal,  but  appear 
quite  opaque  if  of  much  thickness.  These  needles  may 
probably  represent  the  anhydrite  of  gypsum. 

If  calcium  is  present  in  the  ash  as  calcium  sulphate,  it 
will,  of  course,  form  its  characteristic  crystals  if  the  aqueous 
solution  of  the  ash  is  allowed  to  slowly  evaporate. 


Fig.  24.— Crystals  of  calcium  sul 
phate.    After  Haushofer. 


h.  Recognition  of  Calcium  in  the  Cell-sap. 

99.  For  the  recognition  of  calcium  in  the  cell-sap,  Schim- 
per employed  chiefly  the  two  following  reactions: 

I.  On  the  addition  of  ammoniitin  oxalate,  calcium  oxalate 
U  formed  in  the  cells  containing   calcium,  in  the  form  of 


MICROCHEMISTRY.  6/ 

tetragonal   pyramids   at   ordinary  temperatures,  but   in  the 
monoclinic  form  in  a  boiling  solution. 

2.  Fresh  sections  are  placed  directly  in  a  solution  of  a^n- 
rnoniiim  carbonate ;  if  calcium  is  present,  small,  strongly 
doubly  refractive  rhombohedra  of  calcium  carbonate  are 
formed  within  the  cells.  If  the  cell-sap  is  strongly  acid,  it 
should  first  be  neutralized  with  ammonia. 

13.  Mag^nesium,  Mg. 

100.  Schimper  recommends  (II,  214)  the  addition  to  the 
sections  or  to  the  ash,  for  the  recognition  of  magnesium, 
of  a  solution  of  sodium  phosphate  or  of  microcosinic  salt 
{NaNH^HPO,)  reduced  with  a  little  ammonium  chloride. 
There  are  then  formed  rhombic  crystals  of  ammonio-mag- 
nesium  phosphate  (MgNH^PO^)  which  have  in  sections  the 
form  of  coffin-lids,  but  in  the  ash  are  chiefly  the  X-shaped 
skeletons  (cf.  Fig.  20,  §  77). 

Uranyl  acetate  causes,  if  sodium  is  also  present,  the  forma- 
tion of  the  crystals  of  magnesium-sodium-uranyl  acetate, 
already  referred  to  (cf.  §  83). 

101.  Magnesmin  oxalate,  Mg(COOX.  Monteverde  (I) 
found,  in  the  epidermis  of  fresh  leaves  of  Setaria  viridis  and 
in  dried  leaves  of  numerous  PanicecBy  radially  striped  sphae- 
rocrystals  or  irregular  aggregates,  which  probably  consist 
of  magnesium  oxalate.  These  were,  according  to  his  state- 
ments, with  difficulty  soluble  in  water,  insoluble  in  acetic 
acid,  and  soluble  in  hydrochloric,  nitric,  and  sulphnric  acids, 
in  the  latter  without  formation  of  gypsum  needles.  After 
the  addition  of  an  ammoniacal  solution  of  sodium  phosphate 
and  ammonium  chloride,  crystals  of  ammonio-magnesium 
phosphate  were  formed  ;  after  heating,  these  dissolved  with- 
out evolution  of  gas ;  gypsum-water  caused  the  formation  of 
calcium  oxalate  crystals  ;  and  after  treatment  with  caustic 
potash  solution  the  sphaerocrystals  lost  their  striping  and 
double  refraction  and  became  soluble  in  acetic  acid. 

Magnesium  phosphate  (MgO,)3(PO)3?  According  to  Han- 
sen (I,  115),  crystals  of  magnesium  phosphate  are  precipe 
itated  in  the  stem   of  the  sugar-cane  by  alcohol.      These 


68  BOTANICAL   MICROTECHNIQUE. 

have  partly  the  glandular  form  and  are  partly  more  or  less 
regularly  formed  sphaerocrystals.  They  are  soluble  in  cold 
water  with  difficulty,  but  more  easily  so  in  hot  water.  They 
are  also  hardly  soluble  in  acetic  acid,  but  readily  so  in 
mineral  acids,  in  sulphuric  acid  without  the  formation  of 
gypsum  needles.  Ammonium  carbonate  gave  no  precipitate 
with  them,  but  the  ammoniacal  solution  of  ammonium 
chloride  and  sodium  phosphate  produced  a  crystalline  pre- 
cipitate. The  phosphoric  acid  was  recognized  with  ammo- 
nium molybdate  (cf.  §  77,  i). 

14.  Iron,  Fc. 

102.  Weiss  and  Wiesner  (I)  have  shown  microchemically 
that  iron  incrusts  especially  the  thicker  cell-membranes  of 
the  higher  plants  in  the  form  of  insoluble  ferric  and  ferrous 
compounds,  and  that  it  also  occurs  in  the  contents  of  the 
cells.  The  authors  mentioned  used  as  a  reagent  an  alco- 
holic solution  of  potassitim  sulphocyanide  added  directly  to 
sections  cut  with  a  silver  or  platinum  knife.  If  a  red  color 
appears  at  once,  it  shows  the  presence  of  a  soluble  ferric 
compound ;  but  if  it  appears  only  after  the  addition  of  hy- 
drochloric acid,  the  presence  of  a  ferric  compound  insoluble 
in  water  is  shown.  In  the  same  way  sections  were  treated 
with  potassium  sulphocyanide  and  chlorine-water  or  nitric 
acid  to  demonstrate  soluble  or  insoluble  ferrous  compounds. 

Large  quantities  of  iron  compounds  also  occur  as  incrus- 
tations of  the  membrane  in  various  Schizomycetes  {Clado- 
thrixy  Crcnothrix,  etc.)  and  in  Closteritim.  It  also  forms 
thick  crusts,  in  the  form  of  ferric  hydroxide,  on  the  mem- 
branes of  many  ConfervacecB  (cf.  Hanstein  I).  For  the 
microchemical  recognition  of  iron,  a  10^  solution  of  potas- 
sium ferrocyanide,  to  which  a  little  hydrochloric  acid  is 
added,  may  be  used  in  these  cases.  The  reagent  causes 
the  immediate  formation  of  Berlin  blue  in  the  presence  of 
ferric  oxide.  The  presence  of  ferrous  oxide  may  be  recog- 
nized in  the  same  way  by  the  use  of  potassium  ferricyanide.* 

*  A  method  for  the  recognition  of  organically  combined  iron,  the  so- 
called  "  masked  iron,"  has  been  given   by  Molisch  (VII).     But  the  same 


MICROCHEMISTKY.  6^ 

For  Leptothrix  ochracea  Winogradsky  (II,  268)  has  shown 
by  recent  investigations  that  the  iron  is  first  deposited  in 
soluble  form  in  the  gelatinous  envelopes,  most  probably  as. 
a  neutral  ferric  salt  of  an  organic  acid.  This  then  gradually 
passes  over  into  a  basic  salt  insoluble  in  water,  and  finally 
into  almost  pure  ferric  hydroxide,  which  is  transformed  by 
long  submergence  in  water  into  a  modification  somewhat- 
less  soluble  in  hydrochloric  acid. 

B.  Organic  Compounds. 

I.    FATTY   SERIES 
I.  Alcohols. 
Dulcite  (Melampyrite)  {C^lOYi\. 
103.   Dulcite    has   been    recognized    by    Borodin    (I)   by^ 
adding  one  or  a  few  drops  of  alcoJiol  to  sections  of  the  plant- 
under  investigation,  covering  with  a  cover-glass,  and  allow- 
ing them  to   dry  slowly.     Dulcite  then   crystallizes  in  the 
form  of  large  prismatic  or  irregular  flattened  crystals  which 
may  be  distinguished  from  saltpeter  and  asparagin  crystals 
by  being  insoluble  in  a  concentrated  solution  of  dulcite  and 
by  being  transformed  on  heating  to  190°  C.  into  frothy  dark 
brown    masses,    with     complete    decomposition.       Dulcite 
crystals  also  differ  from  the  very  similar  saltpeter  crystals 
in  dissolving  without  color  in  diphenylamine-sulphuric  acid 

(cf.  §  73). 

Suitable  objects  for  study  are  furnished  by  one-year-old 
stems  of  Evonymus  japonicus, 

author  has  recently  shown  (VIII)  that  the  iron  observed  by  him  came  frontv 
the  caustic  potash  used  for  the  reaction,  and  that  therefore  the  results  ob- 
tained by  his  method  are  untrustworthy.  [Carl  Miiller  (I)  has  still  more 
recently  concluded,  not  only  that  Molisch's  proposed  method  is  untrust- 
worthy, but  that  his  explanation  of  the  source  of  the  iron  he  found  is-- 
equally  so.  Miiller  finds  that  the  commercial  hydroxide  in  stick  form  con- 
tains no  iron,  and  that  the  iron  found  in  solutions  of  caustic  potash  comes- 
from  the  glass  of  the  vessels  in  which  they  are  contained.  He  believes  also- 
that  the  "masked"  iron  of  Molisch  is  accumulated  by  plant  specimens  fromi 
the  glass  vessels  in  which  they  are  kept,  and  rejects  Molisch's  view  that  most 
of  the  iron  in  the  plant  is  organically  combined.] 


yo  BOTANICAL   MICROTECHNIQUE. 

2.  Acids. 
a.  Oxalic  Acid  {COOH\ 

104.  For  the  recognition  of  oxalic  acid  and  its  soluble 
salts  Schimper  recommends  (II,  215)  : 

1.  The  addition  of  a  solution  of  calcium  nitrate,  when 
<:rystals  of  calcium  oxalate  are  formed  (cf.  §  99,  i). 

2.  The  addition  of  tiranyl  acetate  causes  the  formation 
of  rhombic  crystals  of  mostly  rectangular  form,  which,  when 
large,  are  plainly  yellow  and  strongly  doubly  refractive,  but 
whose  composition  is  still  unknown. 

3.  Acid  potassiwn  oxalate,  when  present  in  considerable 
quantity,  is  often  directly  recognizable  in  dried  preparations 
by  its  crystalline  form  and  strong  double  refraction  on  com- 
parison with  a  dried  solution  of  the  same  salt,  as  well  as  by 
the  aid  of  Borodin's  method. 

b.   Tartaric  Acid,  C,H,(OH),(COOHX. 

105.  Streng  (III)  has  recommended  for  the  recognition 
of  tartaric  acid  the  addition  of  a  solution  of  barium  chloride 
and  anti7no7iic  oxide  in  hydrochloric  acid.  This  causes  the 
formation  of  rhombic  plates  of  antimonyl-barium  tartrate 
whose  obtuse  angles  measure  128°. 

Schimper  (II,  220)  recommends  the  use  of  the  two  follow- 
ing reactions : 

1.  The  addition  of  potassium  acetate  produces  rhombic- 
hemihedric  crystals  of  the  hardly  soluble  acid  potassium 
tartrate. 

2.  Neutral  solutions  are  treated  with  calciinn  chloride. 
There  are  then  formed  rhombic  crystals  of  calcium  tartrate 
which  represent  chiefly  a  combination  of  an  elongated  prism 
with  the  dome.     Concerning  their  reactions  see  §  95. 

c.  Betuloretic  Acid,  C„H„0,. 

106.  This  acid  is  secreted  by  the  trichome-glands  on  the 
leaves  of  Betula  alba.  It  is  insoluble  in  water,  but  soluble 
in  alcohol,  ether,  alkalies,  alkaline  carbonates,  and  concen- 
trated sulphuric  acid,  in  the  latter  with  a  red  coloration  (cf. 
Behrens  III,  379). 


ICROCHEMISTRY.  'JX 

3.  Fats  and  Fatty  Oils. 

107.  Under  the  names  fats  and  fatty  oils  are  included,  ac- 
cording to  their  consistencies,  the  glycerine  ethers  of  various 
organic  acids  of  high  molecular  weights,  especially  those  of 
palmitic  acid,  C.sHgjCOOH,  stearic  acid,  C^Hj.COOH,  and 
oleic  acid,  C^HgaCOOH. 

But  beside  these,  a  whole  series  of  acids  still  partly  but 
little  studied  have  been  isolated  from  the  various  oils  of 
vegetable  origin  (cf.  Beilstein  I,  427).  An  exact  microchem- 
ical  separation  of  these  compounds  is  not  yet  possible. 
Even  those  reactions  which  should  show  whether  doubtful 
substances  belong  to  the  group  of  fats  still  leave  much  to 
be  desired  in  the  matter  of  exactness,  since  they  nearly  all 
occur  in  the  presence  of  other  substances. 

108.  In  general,  however,  the  fatty  oils  show  the  following 
reactions : 

They  are  insoluble  in  cold  and  hot  water  and  slightly 
soluble  in  alcohol ;  but  castor-oil  forms  an  exception  in 
being  pretty  readily  soluble  in  alcohol. 

They  are  easily  soluble  in  carbon  bisulphide,  ether,  chloro- 
form, petroleum  ether, '^  phenol,  ethereal  oils  (as,  e.g.,  clove- 
oil),  acetone,  and  wood-siprit  (methyl  alcohol). 

According  to  A.  Meyer  (II),  most  fatty  oils  are  insoluble 
m  glacial  acetic  acid,  if  the  quantity  of  acid  is  not  too  great, 
as,  for  instance,  when  the  reaction  is  conducted  under  a 
cover-glass. 

An  aqueous  solution  of  chloral  hydrate  acts  in  the  same 
way  as  glacial  acetic  acid,  according  to  A.  Meyer  (II). 

109.  Alcannin,  the  coloring  matter  contained  in  the  roots 
of  Alcanna  tinctoria,  colors  the  fats  deep  red.  The  solution 
used  for  this  reaction  may  be  prepared  by  dissolving  the 
commercial  alcannin  in  absolute  alcohol,  adding  the  same 
volume  of  water,  and  filtering.  In  this  solution  the  sections 
to  be  tested  are  left  for  one  or  two,  or  better,  six  to  twenty- 
four,  hours.  All  oil-drops  then  appear  deeply  colored ; 
but,  on  the  other  hand,  ethereal  oils  and  resins  show  the 

*[This  is  the  benzinuin  of  the  U.  S.  Pharmacopoeia.] 


72 


BOTANICAL   MICROTECHNIQUE. 


same  reaction.  The  staining  with  alcannin  may  be  much 
hastened  by  warming.  This  is  especially  to  be  recommended 
when  one  has  to  deal  with  fats  which  are  solid  at  ordinary 
temperatures,  as  in  the  cocoa-bean.  If  cross-sections  of 
this  seed  are  heated  to  the  boiling  point  in  a  considerable 
quantity  of  the  above  solution,  the  crystals  of  cocoa-butter 
melt  and  fuse  into  drops,  which  become  colored  deep  red  at 

once. 

110.  Ranvier  has  used  (I,  97)  cyanin  (identical  with 
<hinolin  blue,  bleu  de  quinol^ine)  for  the  recognition  of 
fats.  This  coloring  matter  is  pretty  easily  soluble  in  alco- 
hol, but  practically  insoluble  in  water,  especially  in  cold 
water.  On  the  dilution  of  alcoholic  solutions  with  water, 
precipitates  are  readily  formed,  and  I  have  found  it  most 
-convenient  to  dissolve  the  dye  in  50^  alcohol  and  to  use 
this  solution  directly  for  staining.  Fresh  material  or  such 
as  has  been  fixed  in  any  aqueous  fixing  fluid  (an  aqueous 
solution  of  corrosive  sublimate  or  of  picric  acid,  for  exam- 
ple) may  be  used.  It  is  usually  sufficient  for  the  staining 
to  leave  the  objects  in  the  above  solution  about  half  an 
hour.  Over-stained  sections  may  be  washed  out  with  gly- 
cerine or  concentrated  caustic  potash  solution.  The  per- 
manent preservation  of  these  preparations  in  glycerine- 
gelatine  does  not  appear  to  be  possible ;  at  least,  after  a  few 
months  such  a  preparation  was  completely  decolorized. 

I  can  recommend  as  suitable  objects  for  study  old  leaves 
of  Agave  americana,  which  contain  large  oil-drops  (cf.  §  364) 
in  the  leucoplasts  of  their  epidermal  cells.  These  are 
deeply  colored  in  preparations  made  in  the  way  above 
■described,  while  the  nuclei  and  chromatophores  remain 
unstained.  The  only  disadvantage  of  the  method  consists 
in  the  fact  that  the  lignified  and  suberized  membranes  are 
also  pretty  deeply  stained  by  it. 

111.  Osmic  acid,  commonly  used  in  a  i^  aqueous  solution, 
•colors  most  fats  deep  brown  or  quite  black.  But  this  reac- 
tion, which  depends  on  a  reduction,  may  always  be  checked 
in  a  short  time  by  means  of  hydrogen  peroxide.  According 
to  Flemming  (II),  the  same  thing  may  be  accomplished  with 
oil  of  turpentine,  xylol,  ether,  or  creosote,  but  it  requires 


MICKOCHEMISTR  Y.  J I 

usually  several  hours  and  a  gentle  warming  for  complete 
<iecolorization. 

It  has  been  shown  by  the  researches  of  Altmann  (I,  io6) 
that  this  reaction  is  by  no  means  characteristic  of  all  fats. 
It  is,  on  the  contrary,  suppressed  in  palmitic  acid,  stearic 
acid,  and  their  triglycerides,  in  the  mono-  and  triglycerides 
of  butyrin,  in  lecithin,  jecorin,  and  soap.  But  a  strong 
blackening  occurs  with  free  oleic  acid  and  olein.  These 
two  compounds  are  distinguished  from  each  other  by  the 
fact  that,  when  it  is  blackened  by  osmic  acid,  oleic  acid  is 
still  soluble  in  alcohol,  while  olein  is  not. 

If  tannins  be  present  in  the  cells  to  be  tested  for  fatty 
oils,  they  should  be  extracted  by  boiling  with  water  before 
the  addition  of  the  osmic  acid,  since  they  also  blacken  with 
it.  Ethereal  oils  can  be  removed  by  heating  to  130°  C. 
(cf.  §  145). 

112.  The  saponification  of  fats  under  the  microscope  was 
first  carried  out  by  Molisch  (I,  10,  note).  For  this  purpose 
he  places  the  sections  to  be  studied  in  a  drop  of  a  mixture 
of  equal  parts  of  a  concentrated  solution  of  potassium 
hydrate  and  a  concentrated  solution  of  ammonia.  After 
half  an  hour  or  an  hour  or  an  even  longer  time,  the  oil- 
drops,  "constantly  losing  their  strong  refractive  power, 
harden  into  myelin-like  or  botryoidal  bodies  or  into  irregu- 
lar masses  (soaps)  often  consisting  wholly  of  small  crystal- 
needles." 

Different  objects  seem  to  behave  very  differently  in  this 
respect.  Thus,  I  obtained  very  delicate  crystal-needles 
(cf.  Fig.  25)  on  placing  sections  from 
the  endosperm  of  the  coffee-bean  in  the 
above-mentioned  mixture  of  alkalies. 
These  formed,  after  a  few  hours,  around 
the  oil-drops,  which  in  twenty-four  hours 
were  wholly  converted  into  crystals.  I 
observed  in  places,  within   the  crystal-  Fig.  25.— Oii  -  drops   from 

*  .  the     endosperm    of     the 

aererresfates,  a  strons^ly  refractive  rounded     coffee-bean    five    hours 

°°      o  '  o  -'  after    saponification    with 

body,  which,  as  examination  by  polar-     caustic  potash  and  ammo- 

ized  light  shows,  was  a  sphaerocrystal, 

and  showed  the  familiar  dark  cross,  with  crossed  nicols. 


74  BOTANICAL   MICROTECHNIQUl:. 

In  various  other  objects,  as,  for  instance,  in  sections  from 
the  endosperm  of  Bcrthollctia  excelsa  or  from  the  cotyledons 
of  Hclianthus  anmius,  I  obtained  much  larger  sphaerocrystals 
or  groups  of  them,  which  were  often  not  to  be  distinguished 
from  oil-drops  by  ordinary  illumination,  but  behaved  quite 
like  sphaerites  in  polarized  light. 

Whether  these  differences  are  to  be  attributed  to  chemical 
differences  between  the  various  fats,  or  whether  all  fatty 
oils  yield  crystalline  formations  under  the  treatment  de- 
scribed, must  be  determined  by  further  researches.  It  is 
also  still  to  be  shown  whether  other  compounds,  especially 
many  ethereal  oils,  do  not  show  the  same  relations. 

4.  Wax. 

113.  The  name  wax  is  commonly  given  to  the  substance 
which  covers  those  parts  of  many  plants  which  are  above 
ground  and  gives  them  a  characteristic  bright  blue-green 
color. 

Morphologically,  three  distinct  kinds  of  wax  coverings 
may  be  distinguished.  In  the  first,  the  wax  forms  a  com- 
plete coherent  crust  over  the  epidermis;  in  the  second,  it 
occurs  in  the  form  of  rounded  granules ;  in  the  third,  in  the 
form  of  small  rods. 

Concerning  their  chemical  relations  it  may  be  remarked 
that,  according  to  Weisner's  investigations  (I  and  II),  these 
wax  coverings  contain  true  fats,  free  fatty  acids,  and  a 
number  of  other  substances.  But  in  general  the  study  of 
their  chemical  constitution  has  to  do  with  little  known 
compounds. 

114.  The  wax  coverings  are  characterized  microchemi- 
cally,  as  DeBary  (I,  132)  first  showed,  by  being  always 
insoluble  in  water,  though  they  melt  together  into  drops  in 
boiling  water,  since  their  melting  points  are  all  below  100° 
C.  They  are  also  insoluble  or  hardly  soluble  in  cold  alco- 
hol, but  are  always  completely  dissolved  by  boiling  alcohol. 
In  ether  some  of  them  are  readily  soluble ;  others  are  not 
soluble  or  very  slightly  so.  On  heating  in  a  solution  of 
alcannin  in  50ji^  alcohol,  they  run  together  into  red  drops. 


MICROCHEMISTR  Y, 


/:> 


Since  wax  is  not  wetted  by  water,  the  study  of  the  various 
rods,  granules,  etc.,  is  better  conducted  in  cold  alcohol 
which  wets  the  wax  without  dissolving  it  at  once,  to  say  the 
least. 

115.  The  waxy  incrustations  of  suberized  membranes, 
observed  especially  on  various  epidermal  cells,  as,  for  ex- 
ample, those  of  Aloe  verrucosa,  become  at  once  visible, 
according  to  DeBary  (I),  if  the  sections  are  warmed  under 
a  cover-glass  to  near  the  boiling  point  of  water.  They  then 
separate  from  the  membrane  in  the  form  of  drops.  These 
drops  are  soluble  in  boiling  alcohol  and  behave  chemically 
like  the  wax  coverings  described.  Wax  may  also  be  ex- 
tracted from  the  incrusted  membranes  by  boiling  alcohoL 
The  membranes  always  suffer  in  consequence  a  correspond- 
ing reduction  of  volume,  which  cannot  be  made  good  by- 
subsequent  immersion  in  water. 

5.  Carbohydrates. 

116.  The  carbohydrates  are  characterized,  as  is  well 
known,  by  the  fact  that  they  contain,  besides  carbon,  hy- 
drogen and  oxygen  in  the  same  proportion  as  in  water,  so- 
that  their  general  formula  may  be  written  CxH2yOy. 

Of  course  not  all  organic  compounds  which  show  this- 
empirical  formula  are  included  in  the  carbohydrates,,  and 
already  various  substances  which  were  formerly  included 
here  have  been  transferred  to  other  places  in  the  natural 
system  of  organic  compounds,  after  their  constitution  has; 
become  more  exactly  known.  And  the  recent  investigar- 
tions  of  Emil  Fischer  (I)  have  introduced  a  more  rational 
classification  of  the  carbohydrates. 

But  these  investigations  have  at  present  no  significance 
for  microchemical  methods,  since  the  certain  microscopical 
separation  of  the  compounds  of  this  group  is  as  yet  possible 
only  in  very  rare  cases.  I  will  therefore  restrict  myself 
here  to  the  description  of  microchemical  methods  for  recog- 
nizing some  soluble  carbohydrates.  The  solid  carbohy- 
drates, cellulose  and   its  derivatives,  as  well   as  starch  and 


76  BOTANICAL   MICROTECHNIQUE. 

the  related  compounds,  will  be  discussed  in  Part  III  of  this 
book  (cf.  §§  242-297  and  §§  400-415). 

117.  But  before  we  enter  upon  the  special  reactions  of  the 
soluble  carbohydrates,  two  reactions  common  to  many  car- 
bohydrates may  be  described.  These  were  introduced  into 
microchemistry  by  Molisch  (V),  and  at  first  especially  for 
the  recognition  of  species  of  sugar.  The  reagents  used  are 
rt.naphtol  and  thymol. 

Molisch  uses  a-naphtol  by  treating  sections  not  too  thin, 
on  the  slide,  with  a  drop  of  a  15-20^  alcoholic  solution  of 
the  compound  and  then  adding  two  or  three  drops  of  con- 
centrated sulphuric  acid,  so  that  the  sections  are  wholly 
covered.  In  the  presence  of  cane-sugar,  milk-sugar,  glucose, 
Ixvulose,  maltose,  or  inulin,  the  section  becomes  colored  a 
beautiful  violet  in  a  short  time  (about  two  minutes),  while 
this  reaction  does  not  occur  with  inosite,  mannite,  melam- 
pyrite,  and  quercite. 

If  thymol  be  used  in  the  same  way  intead  of  o'-naphtol,  a 
carmine-red  color  is  produced. 

Concerning  this  reaction  it  should  be  said  that  the  con- 
centrated sulphuric  acid  contained  in  the  reagent  may  split 
off  sugars  from  glucosides,  starch,  cellulose,  and  various 
other  substances,  and  these  may  then  give  the  reaction  in- 
<lirectly.  But  in  the  absence  of  soluble  carbohydrates  the 
reaction  occurs  much  later,  often  only  after  a  quarter  to  a 
half  of  an  hour.  Then,  too,  according  to  Molisch,  one  may 
reach  a  definite  conclusion  as  to  the  presence  of  soluble  car- 
bohydrates by  treating  in  the  same  way  a  fresh  section  and 
one  extracted  with  boiling  water.  If  the  reaction  occurs 
markedly  sooner  in  the  former,  it  is  proved  that  it  depends 
.upon  the  presence  of  soluble  substances. 

But  it  has  been  shown  by  Nickel  (I,  31)  that,  besides  the 
compounds  mentioned,  a  number  of  bodies  of  wholly  differ- 
ent constitution  give  the  same  reaction,  especially  proteids, 
kreatin,  and  vanillin.  According  to  Nickel,  it  is  very  prob- 
able that  these  reactions  depend  upon  the  fact  that  the  sul- 
phuric acid  splits  off  furfurol  from  the  compounds  named. 


MICR  O  CHEMIS  TR  Y.  7/ 

a.  Glucose,  CgHj^Oe. 

118.  In  botanical  literature  the  name  glucose  commonly 
includes  all  those  kinds  of  sugar  which  precipitate  cuprous 
oxide  from  an  alkaline  solution  of  copper.  In  most  cases 
we  have  undoubtedly  to  do  with  the  compound  known  to 
chemists  as  glucose  (grape-sugar  or  dextrose)  ;  but  there  are 
many  other  substances,  as,  for  instance,  laevulose,  lactose, 
and  many  glucosides,  which  give  the  same  reaction  ;  and 
great  care  should  be  exercised  as  to  the  significance  of  the 
copper-reaction,  where  such  other  compounds  are  not  ex- 
cluded. 

The  reaction  named  is  best  conducted,  according  to  A. 
Meyer  (IV),  by  first  placing  sections,  from  two  to  four  cells 
in  thickness,  of  the  object  to  be  studied,  in  a  concentrated 
aqueous  solution  of  aipric  sulphate  for  a  short  time,  then 
washing  them  in  distilled  water  and  finally  placing  them  in 
a  boiling  solution  of  lo  grams  of  Rochelle  salt  and  lo  grams 
Gi  potassium  hydrate  in  lo  grams  of  water.  There  are  then 
precipitated  in  the  cells  containing  glucose,  vermilion  gran- 
ules of  cuprous  oxide,  whose  color  may  best  be  seen  by  dark 
ground  illumination,  as  they  often  appear  almost  wholly 
black  by  transmitted  light,  especially  with  a  narrow  cone  of 
rays.  Cuprous  oxide  remains  at  first  unchanged  in  glycer- 
ine, even  on  boiling  ;  but,  according  to  A.  Fischer  (V,  74),  it 
is  dissolved  after  some  weeks  by  glycerine,  as  well  as  by 
Canada  balsam. 

119.  The  reaction  may  also  be  conducted  on  the  slide  in 
the  manner  recommended  by  Schimper  (cf.  Strasburger  I, 
73),  by  warming  the  sections,  which  should  not  be  too  thin, 
under  a  cover-glass  in  a  drop  of  Fehling's  solution  *  until 
little  bubbles  begin  to  be  formed.  Stronger  heating  usually 
-causes  marked  changes  in  the  cell  contents. 

*  Fehling's  solution  may  be  prepared,  according  to  Dragendortf  (I,  70), 
by  making  three  different  solutions  containing  respectively,  in  a  liter  of 
water,  35  grams  of  cupric  sulphate,  173  grams  of  Rochelle  salt  (sodium- 
potassium  tartrate,  NaK(COO)2C2H2(OH)2 +  4H2O),  and  120  grams  of 
caustic  soda.  Just  before  use,  a  mixture  of  one  volume  of  each  of  these 
solutions  is  added  to  two  volumes  of  water.  This  mixture  becomes  changed 
in  time,  while  the  separate  solutions  may  be  kept  indefinitely. 


78  BOTANICAL   MICROTECHNIQUE. 

120.  To  recognize  glucose  in  vessels,  A.  Fischer  (V,  74) 
placed  suitable  pieces  from  branches  split  through  the 
middle  in  a  concentrated  aqueous  solution  of  cupric  sul- 
phate for  five  minutes,  then  rinsed  them  in  water  and. placed 
them  in  a  boiling  solution  of  Rochelle  salt  and  caustic  soda,. 
in  which  he  let  them  boil  for  from  two  to  five  minutes. 
The  cuprous  oxide  is  then  precipitated  in  the  cells  which 
formerly  contained  sugar,  and  the  wood  may  be  readily  cut. 
Dried  wood  and  alcoholic  material  in  large  pieces,  whose 
old  surfaces  have  been  previously  removed,  may  serve  for 
the  reaction. 

b.  Canc-sngary  Saccharose^  Cj^H^O,,. 

121.  Cane-sugar  is  widely  distributed  among  plants,  and 
suitable  material  for  study  is  afforded  by  pieces  of  a  sugar- 
beet.  Even  on  gentle  boiling  it  cannot  precipitate  cuprous 
oxide  from  Fehling's  solution  ;  but  on  longer  boiling  in  this 
solution,  the  cane-sugar  becomes  converted,  in  consequence 
of  the  strongly  alkaline  reaction  of  the  solution,  into  the 
so-called  invert-sugar,  a  mixture  of  glucose  and  laevulose, 
which  reduces  Fehling's  solution. 

For  the  microchemical  recognition  of  cane-sugar,  sections 
not  too  thick  are  placed,  according  to  Sachs  (I,  187),  for  a 
short  time  in  a  concentrated  aqueous  solution  of  cupric  sul- 
phate, rapidly  rinsed  in  water,  and  then  transferred  to  a 
solution  of  one  part  potassium  hydrate  in  one  part  water, 
heated  to  boiling.  If  cane-sugar  is  present,  there  appears 
in  the  cells  containing  it  a  sky-blue  color  which  gradually 
diffuses  into  the  potash.  A  careful  microscopic  control  of 
this  reaction  is  recommended,  since,  as  Sachs  states,  the 
young  cell-membranes  often  become  colored  deep  blue 
under  the  same  treatment. 

Fehlings  solution  may  also  be  used  for  the  recognition  of 
cane-sugar,  which  gives  a  blue  solution  with  it  also.  For 
the  method  of  using  it,  see  §  119. 

c.  Inulin,  C.,H,„0., 
122.   Inulin  is  pretty  readily  soluble  in  water  and  occurs 
dissolved  in  the  cell-sap  of  many  plants.     But,  since  it  is 


MICK  0  CHE  MIS  TR  Y,  79 

insoluble  in  alcohol  and  glycerine,  it  is  precipitated  in  the 
form  of  well  developed  sphaerocrystals  which  often  fill  large 
cell-masses,  when  parts  of  plants  containing  inulin  are  pre- 
served for  a  time  in  one  of  these  fluids.  Such  sphaerocrys- 
tals may  be  observed  on  examining  larger  parts  of  plants, 
as,  for  instance,  halved  Dahlia  roots,  which  have  lain  several 
weeks,  or  longer,  in  alcohol  or  concentrated  glycerine. 
After  being  kept  for  years  in  alcohol  these  are  with  diffi- 
culty soluble  in  cold  water,  as  Leitgeb  (I,  136)  has  shown  ; 
while  those  in  alcoholic  material  a  few  weeks  old  dissolve 
pretty  readily  in  it.  But  in  hot  water  even  old  inulin  sphae- 
rites  are  easily  soluble,  so  that  they  are  readily  distinguished 
from  the  otherwise  similar  sphaerites  of  calcium  phosphate 
(cf.  §  96).  If  sections  from  alcoholic  material  of  Dahlia 
roots,  which  always  contain  calcium  phosphate  sphaerites 
with  those  of  inulin,  especially  near  the  cut  surfaces  of  the 
roots,  be  heated  on  the  slide  in  water  to  the  boiling  point, 
the  inulin  sphaerites  dissolve  almost  instantly,  provided  the 
sections  are  not  too  thick,  while  the  sphaerocrystals  of  cal- 
cium phosphate  remain  for  a  time  quite  unchanged. 

The  inulin  sphaerites  are  also  soluble  easily  and  without 
residue  in  concentrated  sulphuric  acid,  while  those  of  calcium 
phosphate  are  transformed  by  it  into  gypsum  (cf.  §  96). 

Fehling's  solution  is  not  directly  reduced  by  inulin. 

123.  For  the  rapid  recognition  of  inulin,  the  reagents  for 
sugar  recommended  by  Molisch  (V,  918)  may  be  used  (cf. 
§  117).  Thus  the  sphaerocrystals  of  inulin  dissolve  with  a 
very  deep  violet  color,  if  the  sections  containing  them  are 
treated  with  a  10^  alcoholic  solution  of  a-naphtol,  then  with 
a  few  drops  of  concentrated  sulphuric  acid,  and  are  then 
slightly  warmed  under  a  cover-glass. 

But  if  thymol  is  added  in  the  same  way,  a  red  color  ap- 
pears, according  to  Molisch. 

Green  has  recommended  (I)  orcin  as  a  reagent  for  inulin. 
The  sections  to  be  studied  are  saturated  with  an  alcoholic 
solution  of  orcin  and  boiled  in  hydrochloric  acid.  A  deep 
orange-red  color  then  appears  if  inulin   is   present.     Any 


8o  fiOTAA'/CAL   MICROTECHNIQUE. 

sphserocrystals  of  inulin  are,  naturally,   dissolved,  and  the 
spaces  occupied  by  them  appear  red. 

\[ phlorogliicin  is  used  instead  of  orcin,  the  color  produced 
is  browner. 

d.  Glycogen,  CgH,„Oj. 

124.  Glycogen,  which,  according  to  Errera's  investiga- 
tions, is  very  widely  distributed  in  the  cells  of  fungi,  is  char- 
acterized, as  he  has  shown,  by  forming  a  colorless  and 
strongly  refractive  substance  within  the  living  cells,  and  by 
becoming  colored  a  deep  red-brown  with  a  solution  of 
iodine  and  potassium  iodide.  This  color  disappears  on  warm- 
ing to  50°  or  60°  C,  and  reappears  on  cooling.  The  gly- 
cogen dissolves  in  water  if  the  preparation  is  crushed 
(Errera  I). 

Since  the  intensity  of  the  color  produced  by  iodine  de- 
pends on  the  amount  of  glycogen  present,  by  the  use  of 
iodine  solutions  of  a  given  strength  one  may  obtain  some 
quantitative  estimate  of  the  glycogen  from  the  color.  For 
this  purpose  Errera  (II)  places  the  objects  to  be  studied 
directly  in  a  solution  containing  45  grams  of  water,  .3  gram 
of  potassium  iodide,  and  .1  gram  of  iodine.  If  the  glycogen 
is  present  in  extremely  small  quantity,  the  color  will  be 
orange  rather  than  brown.  Then  a  somewhat  more  con- 
centrated solution  (i  :  lOo)  may  be  used  ;  but  it  must  be 
used  very  carefully. 

c.  Dextrine,  Cj^H^gOjo. 

125.  Dextrine  is  the  name  given  to  the  transition  product 
between  starch  and  maltose.  It  is  distinguished  from  the 
latter  by  being  insoluble  in  84%  alcohol.  Therefore  Sachs 
(I,  187)  proposed,  for  the  microchemical  recognition  of 
dextrine,  that  sections  of  plants  which  showed  the  presence 
of  copper-reducing  substances  with  Fehling's  solution  should 
be  placed  in  95^^  alcohol  for  10  to  24  hours  to  completely 
dissolve  out  the  glucose.  If  the  copper-reaction  then  still 
took  place,  he  deduced  the  presence  of  dextrine.  But, 
according  to  more  recent  investigations,  pure  dextrine  can- 


MICROCHEMIS TR  V.  51 

not  reduce  Fehling's  solution,  and  it  is  also  doubtful 
whether  this  substance  occurs  in  recognizable  quantity 
within  the  plant  (cf.  Beilstein,  I,  883). 

6.  Sulphur  Compounds. 

126.  Of  sulphur  compounds  of  the  fat  group  only  oil  of 
garlic  and  oil  of  mustard  have  been  studied  with  reference 
to  their  microchemical  recognition. 

a.   Garlic  Oil,  Allyl  Sulphide,  (C3H  J,S. 

Garlic  oil,  which  occurs  in  almost  all  parts  of  the  various 
species  of  Allium,  gives  the  following  reactions: 

With  platinum  chloride,  a  characteristic  yellow  precipitate  ; 

With  mercury  salts,  a  white  precipitate ; 

With  palladous  nitrate,  a  kermes-brown  precipitate  ; 

With  a  \-2%  solution  of  silver  nitrate,  a  finely  granular 
precipitate  of  silver  sulphide  ; 

With  concentrated  sulplmric  acid,  a  beautiful  red  color ; 

With  gold  chloride,  a  yellow  precipitate. 

For  microchemical  purposes  the  palladous  nitrate  and 
silver  nitrate  serve  best,  according  to  Voigt  (I),  and  it  is 
convenient  to  place  large  portions  of  the  plants  in  the 
solution  and  to  hasten  its  penetration  by  the  aid  of  the  air- 
pump.     After  hardening  in  alcohol,  sections  may  be  cut. 

b.  Mustard-oils,  Alkylthiocarbimides,  SC  :  N-R. 

127.  The  mustard-oils  include  a  group  of  homologous 
compounds  which  contain  the  atom  group  SC  :  N  and  an 
alkyl  radical.  The  best  known  of  them  is  allylic  mustard- 
oil  (allyl  sulphocyanate,  CgH^CNS),  which  is  separated  by 
the  ferment  myrosin  from  potassium  myronate,  which  be- 
longs to  the  glucosides  and  occurs  especially  in  the  seeds  of 
black  mustard.  The  reactions  proposed  by  Solla  (II)  for 
the  microchemical  recognition  of  allyl  mustard-oil  have 
proved  useless  on  being  tested  by  Bachmann  (VI)  and 
Molisch  (I,  33),  so  that  we  have  at  present  no  special  re- 
action for  those  bodies  which  is  microchemically  applicable. 


^2  BOTAMCAL   MICROTECHNIQUE, 

7.  Araido-compounds. 

128.  The  amido-compounds  (amido-acids  and  acid  amides) 
are  characterized  by  the  fact  that  they  contain  the  uni- 
valent radical  NH,.  They  are  therefore  nitrogenous,  and 
very  probably  play  a  most  important  role  in  the  formation 
and  transference  of  the  albuminous  materials.  We  have, 
however,  trustworthy  microchemical  methods  for  recogniz- 
ing only  two  of  the  amido-compounds  of  the  fat  group, 
leiicin  and  asparagin ;  while  of  the  aromatic  amido-com- 
pounds tyrosin  may  be  mentioned  here  (cf.  §  1 34). 

a.  Li'ucin,  Amido-caproic  Acid,  CjHjoNHj.COOH. 

129.  Leucin  was  microchemically  recognized  by  Borodin 
(IV)  in  the  leaves  of  etiolated  specimens  of  Paspahnn  elegans 
and  Dahlia  variabilis.  He  made  use  for  this  purpose  of  its 
property  of  subliming  without  decomposition  when  care- 
fully  heated  to  170°  C.  Dried  sections  were  covered  on  the 
slide  with  a  clean  cover-glass  and  carefully  heated.  When 
leucin  was  present,  tiny,  crystalline,  doubly  refractive  scales 
of  this  compound  were  deposited  on  the  under  side  of  the 
cover-glass,  under  this  treatment.  These  could  then  be 
tested  with  a  saturated  aqueous  solution  of  leucin  (cf.  § 
71,  note). 

Leucin  is  also  deposited  in  crystalline  form,  as  are  aspara- 
gin and  tyrosin,  if  sections  containing  it  are  treated  with 
alcohol  and  allowed  to  dry  slowly  under  a  cover-glass.* 

h.  Asparagin,  Amide  of  Asp  art  ic  {Amidosticcinic)  Acid, 
C,H3NH,.CONH,.COOH. 

130.  Asparagin  is  soluble  in  water  and  occurs  only  in 
solution,  in  vegetable  cells.  For  the  recognition  of  aspar- 
agin according  to  the  method  first  used  by  Borodin  (II),  the 
sections  are  treated  with   absolute  alcohol  under  a  cover- 


*  Leucin  has  recently  been  recognized  by  Belzung  (II)  in  the  seedlings  of 
J.upinus  alhus.  It  is  here  precipitated  in  the  tissues  preserved  in  glycerine 
in  the  form  of  heart-shaped  lamellae,  which  are  often  aggregated  into 
sphaerocrystals. 


MICROCHEMISTRY.  83 

glass  and  then  the  preparation  is  allowed  to  dry  slowly. 
The  asparagin  then  separates  out  in  crystals,  among  which 
rhombic  plates  with  an  obtuse  angle  of  129°  18'  are  espe- 
cially characteristic.  This  angle  measures  in  the  otherwise 
similar  crystals  of  potassium  nitrate  99°  44^"^     A  positive 

o  o 

Fig.  26.— Crystals  of  asparagin.  Fig.  27.— Crystals  of  saltpeter, 

decision  between  asparagin  and  saltpeter  is  therefore  usually 
possible  without  the  actual  measurement  of  angles,  after 
some  practice  (cf.  Figs.  26  and  27).  The  observation  of  the 
crystals  is  much  aided  by  polarized  light. 

According  to  the  method  already  employed  by  C.  O. 
Miiller  (I),  a  solution  of  dipJienylanmie  may  be  used  to  dis- 
tinguish between  asparagin  and  saltpeter,  since  the  former 
is  dissolved  without  color,  while  saltpeter  crystals  produce  a 
deep  blue  color  with  it  (cf.  §  J^).  The  behavior  of  asparagin 
crystals  on  heating  is  also  characteristic.  At  about  100°  C. 
they  are  dissolved  by  their  water  of  crystallization,  while  at 
about  200°  C.  transformation  into  brown  drops  of  froth  takes 
place.  Finally,  doubtful  crystals  may  be  tested,  after  Boro- 
din's method  (cf.  §  71,  note),  with  a  saturated  aqueous  solu- 
tion of  asparagin. 

According  to  Leitgeb  (II,  222),  the  crystallization  of 
asparagin  is  hindered  by  the  presence  of  inulin,  as  well  as 
of  gum,  sugar,  glycerine,  and  other  viscous  fluids.  This 
author  was,  however,  able  to  recognize  asparagin  in  Dahlia 
roots,  which  are  rich  in  inulin,  after  placing  transverse  slices 
of  them  a  centimeter  thick  in  90^  alcohol.  After  a  few  days 
he  saw  on  the  cut  surfaces  well  formed  crystals,  which,  after 
being  as  completely  freed  from  inulin  as  possible,  showed 
the  above  described  asparagin  reactions. 

*  But  angles  of  109°  56'  and  118°  50'  are  also  possible  (cf.  C.  O.  Miiller 
I,  15);  I  have,  however,  never  observed  these  angles,  even  after  a  very- 
slow  crystallization  of  pure  potassium  nitrate. 


84  BOTANICAL    MICROTECHNIQUE. 

II.  AROMATIC   SERIES. 

I.  Phenols. 

a.  Eugenol,  C.H3.OH.OCH3.C3H,. 

131.  Molisch  (I,  40  and  44)  recommends  caustic  potash  for 
recognizing  eugenol,  which  occurs  in  the  oils  of  pimento  and 
clove ;  and  he  places  the  sections  in  a  completely  saturated 
solution  of  potassium  hydroxide.  There  are  then  formed  in 
a  short  time  (about  five  minutes),  from  each  oil-drop,  numer- 
ous, often  very  long,  columnar  or  needle-shaped,  colorless 
crystals  of  potassium  caryophyllate.  Sections  of  cloves  are 
especially  recommended  as  suitable  objects  for  their  study. 

The  reactions  with  concentrated  sulphuric  or  nitric  acid 
are  less  trustworthy.  The  former  first  colors  eugenol  yellow- 
ish and  then  at  once  deep  blue,  with  a  tint  of  violet  after  a 
time,  and  finally  brown.  The  latter  colors  it  brilliant  orange 
or  brown-red. 

b.  Phlorogluciu,  C^H,(0H)3. 

132.  Phloroglucin  occurs  in  the  living  cell  only  in  solu- 
tion in  the  cell-sap.  It  is  best  recognized  by  means  of  the 
mixture  of  vanillin  and  hydrochloric  acid  proposed  by  Lindt 
(I).  This  is  prepared  by  dissolving  .005  gram  of  vanillin  in 
.5  gram  of  alcohol  and  then  adding  .5  gram  of  water  and  3 
grams  of  concentrated  hydrochloric  acid.  It  is  best  to  apply 
this  reagent  to  previously  dried  sections ;  when,  if  phloro- 
glucin be  present,  a  light  red  color  is  produced,  which  later 
becomes  somewhat  violet-red.  Orcin  gives  a  bright  blue 
color  with  a  red  shading  ;  and  many  other  related  substances 
give  no  color. 

Very  small  quantities  of  phloroglucin  are  not  recognizable, 
on  account  of  the  red  color  of  lignified  membranes  pro- 
duced by  the  addition  of  hydrochloric  acid  (cf.  §§  255  and 
257). 

According  to  Waage  (I,  253),  phloroglucin  in  the  living 
cell  takes  up  methylene  blue  as  do  the  tannins. 


MICROCHEMIS TR  V.  8  J 

c.  Asaron,  CeH,.(OCH3)3.C3H,. 

133.  Asaron  occurs  especially  in  the  rhizome  and  the 
root  of  Asartim  europceum  and  is  here  dissolved  in  what  is 
very  probably  an  ethereal  oil,  which  almost  entirely  fills  many 
parenchymatous  cells  of  the  bark.  It  is  distilled  over  with 
water  vapor  and  forms  colorless  crystals  which  are  insoluble 
in  water,  but  readily  soluble  in  alcohol,  ether,  chloroform, 
and  acetic  acid. 

For  its  microchemical  recognition  Borscow  (I,  18)  recom- 
mends especially  concentrated  sulphuric  acidy  which  colors  it 
first  yellow,  and  later  orange.  The  oil-drops  containing 
asaron,  which  are  found  in  fresh  sections,  give  the  same 
reaction. 

2.  Acids. 

a.    Tyrosin,  p-Oxyphenyl-a-ainidopropionic  Acidy 
CeH,OH.CH,.CHNH,.COOH. 

134.  Tyrosin  has  been  microchemically  recognized  in  vari- 
ous parts  of  plants,  especially  by  Borodin  (II,  816  and  III,. 
591).  He  proceeded  by  treating  sections  of  the  objects  to 
be  tested  with  alcohol  and  then  allowing  them  to  dry  slowly. 
Tyrosin  then  crystallizes  out  in  dendritic  or  tufted  groups, 
of, strongly  refractive  needles. 

These  are  rather  slightly  soluble  In  water  (in  2454  parts  at 
20°  C,  in  154  parts  at  100°  C,  according  to  Beilstein,  11^ 
1006) ;  and  they  are  of  course  insoluble  in  a  concentrated 
aqueous  solution  of  tyrosin;  but  Borodin's  method  (§71^ 
note)  is  difificult  to  use  with  precision  in  this  case,  on  account 
of  the  slight  solubility  of  tyrosin.  It  is,  however,  character- 
istic of  tyrosin  crystals  that  they  are  colored  deep  red  by 
Millons  reagent.  They  also  leave  a  yellow  residue  when 
they  are  carefully  evaporated  with  nitric  acid.  From  this 
arises  a  deep  red-yellow  fluid,  on  the  addition  of  caustic 
soda;  and,  after  it  dries,  a  red-brown  crystalline  deposit 
remains  (Leitgeb  II,  229). 

135.  According  to  Leitgeb  (II)  the  crystalHzation  of  tyro- 
sin is  more  or  less  completely  prevented  by  the  presence  of 


g6  BOTANICAL   MICROTECHNIQUE. 

inulin,  in  proportion  to  its  abundance.  But  this  author  suc- 
ceeded in  obtaining  from  Dahlia  roots  large  crystal-aggre- 
gates of  tyrosin  by  placing  a  transversely  halved  root  in  a 
cylindrical  vessel  with  the  cut  surface  upward,  and  then  fill- 
ing the  vessel  with  alcohol  until  about  a  third  of  the  root 
projected  above  the  fluid.  The  tyrosin  then  usually  crys- 
tallized on  the  cut  surface  in  a  few  days. 

b.  Ellagic  Acid,  C„H,0,+2H,0. 

136.  G.  Gibelli  (I)  found  in  the  stem  and  root  of  chestnut- 
trees  attacked  by  the  ''  malattia  dell'  inchiostro"  [ink-dis- 
ease], sphaerocrystals  of  ellagic  acid.  These  were  soluble 
in  water  and  alkalies,  and  dissolved  in  potassium  carbonate 
•with  a  yellow  color,  in  concentrated  nitric  acid  with  a  gar- 
net-red color.  Ferric  chloride  produced  a  greenish-black 
color,  and  silver  nitrate  a  red-brown  one. 

3.  Aldehydes. 
Vanillin,  C.H3.OH.OCH3.CHO. 

137.  Vanillin  occurs  often  on  the  surface  of  dried  Vanilla 
pods  in  the  crystalline  form  ;  but  in  their  interior  it  is  found 
only  dissolved  or  in  the  amorphous  condition.  It  is  readily 
soluble  in  ether  and  alcohol,  hardly  soluble  in  cold  water, 
and  more  readily  so  in  hot  water.  It  is  colored  blue  by 
ferric  chloride,  but  this  reaction  cannot  be  used  microchem- 
ically,  according  to  MoHsch  (I,  47).  Besides,  vanillin  gives 
characteristic  color-reactions  with  numerous  organic  com- 
pounds. It  gives  a  brick-red  color  with  phloroglucin  or 
rcsorcin  and  sulphuric  acid,  a  red-violet  with  phloroglucin 
and  hydrochloric  acid,  yellow  with  aniline  sulphate,  red  with 
orcin  and  hydrochloric  acid,  yellow  with  metadiainidobenzol, 
carmine-red  with  thymol,  hydrochloric  acid  and  potassium 
chlorate  (cf.  §  254). 

Of  these  reagents  the  best  adapted  to  microchemical  use 
are,  according  to  Molisch,  orcin  and  phloroglucin.  The 
first  may  be  used  conveniently  in  a  4%  solution,  the  sections 
to  be  tested  being  wet  with  it   on  the  slide  and  then  treated 


MICR  0  CHE  MIS  TRY.  '^f 

with  a  large  drop  of  concentrated  sulphuric  acid.  If  vanillin 
be  present,  the  section  at  once  becomes  colored  deep  car- 
mine-red throughout  its  whole  extent.  If  phloroglucin  is 
used  in  the  same  way,  a  brick-red  color  is  instantly  ob- 
tained with  sulphuric  acid  ;  but  the  color  produced  by  orcin 
is  even  more  striking. 

4.  Quinones. 

138.  The  quinones  are  characterized  by  the  fact  that  the 
two  para-atoms  of  hydrogen  in  the  benzol  molecule  are  re- 
placed by  two  atoms  of  oxygen  which  are  either  united 
together  by  their  second  valence,  or  to  the  carbon  atoms 
concerned  by  both  valences.  They  are  mostly  colored  deep 
yellow.  It  is  not  very  probable,  from  the  investigations 
already  made,  that  they  play  a  very  important  role  in  the 
chemistry  of  the  plant.  Nucin,  emodin,  and  chrysophanic 
acid  have  been  recognized  microchemically. 

a.  Jiiglon,  Nucin,  Oxynaphtoquinone,  C,oHgO.,.OH. 

139.  Nucin  has  been  recognized  in  the  cell-sap  of  the 
parenchyma  of  the  outer  husk  (pericarp)  of  the  fruit  of  Jug- 
lans  regia  by  O.  Herrmann  (I,  183).  He  used  for  this  pur- 
pose a  solution  of  ammonia,  or  better,  the  fumes  of  ammo- 
nia, which  at  first  color  the  nucin  a  brilliant  purple  ;  but 
this  color  gradually  passes  into  brown. 

b.  Emodin,    Trioxymethylanthraquinone,  C,4H^02.CH3.(OH)3, 

140.  Bachmann  (I)  observed  in  the  lichen  Nephroma  hisi- 
tanica  that  small  yellow  crystalline  granules  adhere  to  the 
exterior  of  the  hyphae  of  the  pith,  which  agree  in  their 
microchemical  reactions  with  the  emodin  previously  pro- 
duced macrochemically  only  from  the  rhubarb  root  and 
the  fruits  of  Rhamnus  frangida.  They  are  dissolved,  like 
chrysophanic  acid,  by  caustic  potash  and  soda  solutions  with 
a  red  color.  Lime  and  baryta  waters  color  them  dark  red 
but;  do  not  dissolve  them.  But  they  are  distinguished  from 
chrysophanic  acid  by  being  readily  soluble  in  alcohol,  glacial 


2S  BOTANICAL   MICROTECHNIQUE. 

acetic  acid,  and  amyl alcohol,  and  in  dissolving  in  concentrated 
sulphuric  acid  with  a  saffron-yellow  color ;  while  chryso- 
phanic  acid  is  very  slightly  soluble  in  the  first  three  reagents 
and  dissolves  in  sulphuric  acid  with  a  rose-red  color.  Ac- 
cording to  Fr.  Schwarzdl,  25i),emodin  is  also  distinguished 
from  chrysophanic  acid  by  dissolving  in  ammonium  carbojiate 
with  a  red  color,  while  the  latter  remains  unchanged  in  this 
reagent. 

c.  Chrysophaiiic  Acid,  C,,H,0,.CHg.(OH),. 

141.  Chrysophanic  acid  has  been  observed  especially  in 
the  lichen  Physcia  parictina  and  in  the  roots  of  various  Poly- 
gonacece.  In  the  latter  it  occurs,  according  to  Borscow  (I), 
in  the  form  of  yellow  granules  imbedded  in  the  cytoplasm. 
But  in  Physcia  it  is  in  the  form  of  crystalline  granules  ad- 
hering to  the  membrane,  as  Fr.  Schwarz  (II,  262)  has  shown, 
in  opposition  to  Borscow  (I).  These  are  very  small  and  can 
be  plainly  recognized  only  by  strong  magnification.  In 
polarized  light  they  glisten  brightly  with  crossed  nicols. 
They  are  characterized  by  the  deep  crimson  color  which 
they  take  with  caustic  potash  or  ammonia ;  while  lime  and 
baryta  waters  color  them  dark  red,  without  dissolving  them 
{see  also  §  140). 

5.  Hydrocarbons,  (CioHi8)x. 

142.  A  large  group  of  various  vegetable  substances  is 
included  by  Beilstein  (III,  279)  under  this  title.  They  repre- 
sent either  terpenes  of  the  composition  C,oH,g  or  polymeri- 
zation products  of  these,  or  at  least  are  very  nearly  related 
to  them.  Beside  the  true  terpenes  there  are  included  here 
■especially  the  ethereal  oils,  caoutchouc  and  gutta  percha, 
the  resins  and  balsams.  The  chemical  constitution  of  some 
of  these  compounds  has  been  very  little  studied,  and  doubt- 
Jess  many  of  the  substances  placed  here  will  in  time  be 
transferred  to  other  parts  of  the  natural  system,  especially 
many  of  the  so-called  ethereal  oils.  Thus,  the  chief  constit- 
uent of  the  so-called  ethereal  oil  of  the  species  of  Allium^ 


MICROCHEMISTRY.  89 

garlic-oil,  is  a  relatively  simple  compound,  allyl  sulphide  (cf. 
§  126). 

Since  we  have  no  microchemically  applicable  reactions  for 
most  of  these  compounds,  one  must  be  content  in  most 
cases  to  recognize  them  as  members  of  this  group,  which 
may  conveniently  be  called  the  group  of  the  terpene-like 
compounds,  except  where  macroscopic  studies  of  the  sub- 
stances contained  in  the  plant  concerned  afford  points  of 
vantage  which  may  be  turned  to  account  microchemically. 
In  many  cases  even  this  recognition  cannot  be  made  with 
certainty,  for  lack  of  a  completely  trustworthy  reaction  for 
the  group. 

143.  If  we  omit  caoutchouc  and  gutta  percha,  most  of 
these  compounds  are  characterized  by  being  strongly  refrac- 
tive and  quite  or  almost  insoluble  in  water.  They  are,  how- 
ever, soluble  in  the  solvents  of  fats,  like  ether,  chloroform, 
carbon  bisulphide,  benzol,  and  ethereal  oils.  They  are  also 
mostly  readily  soluble  in  cold  alcohol.  Like  the  fats,  they 
are  deeply  colored  by  alcannin^  and  the  process  is  just  the 
same  as  for  the  fatty  oils  (§  109). 

Besides  these,  the  following  special  reactions  may  be 
described  : 

a.  Ethereal  Oils. 

144.  The  ethereal  oils  are  characterized  microchemically 
by  the  fact  that  they  may  be  obtained  from  the  plants  by 
distillation  with  water  vapor,  and  that  they  leave  on  paper 
a  greasy  spot  which  disappears  after  a  time ;  and  for  their 
microchemical  recognition  their  volatility  may  be  utilized. 
To  test  this,  sections  of  the  parts  concerned  may  be  boiled 
in  water  for  a  time  and  examined  with  the  microscope  before 
and  after  the  boiling. 

According  to  A.  Mayer  (II),  it  is  sufficient  for  the  removal 
of  all  ethereal  oils  to  heat  the  uncovered  sections  for  ten 
minutes  in  a  drying  oven  up  to  130°  C,  since  the  fatty  oils 
remain  unchanged  under  this  treatment.  Otherwise  the 
ethereal  oils  agree  with  the  fatty  oils  in  being  browned  or 


go  BOTANICAL   MICROTECHNIQUE. 

blackened  by  osmic  acid  and  deeply  stained  by  alcannin  or 
iyamn{Q,[.  §§109-111). 

Most  ethereal  oils  are  also  readily  soluble  in  glacial  acetic 
acid  and  in  an  aqueous  solution  of  chloral  hydrate. 

[Mesnard  (I)  has  used  the  following  method  for  distin- 
guishing the  ethereal  from  the  fatty  oils.  He  cemented  two 
glass  rings  of  different  sizes  concentrically  to  a  slide,  the 
inner  ring  being  also  lower  than  the  outer.  The  space  be- 
tween the  two  rings  was  filled  with  strong  hydrochloric  acid, 
and  the  sections  to  be  examined  were  placed  on  the  under 
surface  of  a  cover-glass  resting  on  the  outer  ring,  in  a  hang- 
ing drop  of  glycerine  containing  a  large  proportion  of  sugar. 
Other  sections  may  be  placed,  for  longer  exposure,  on  a 
small  cover  that  rests  on  the  inner  ring.  In  this  apparatus 
the  HCl  is  taken  up  with  water  by  the  glycerine,  and,  after 
a  time,  the  ethereal  oils  present  exude  in  golden-yellow 
drops,  which  later  disappear.  This  exudation  in  drops  never 
occurs  with  fatty  oils.] 

It  may  be  observed  here  that  J.  Behrens  (I)  has  seen  on 
the  glandular  hairs  of  Ononis  spinosa  a  strongly  refractive 
secretion  which  became  colored  deep  red,  even  in  very 
dilute  aqueous  solutions  of  fuchsin.  Nothing  definite  can 
be  said  as  to  the  chemical  composition  of  this  secretion,  but 
it  is  probable  that  it  belongs  in  the  category  of  ethereal 
oils. 

h.  Resins  and  Terpenes. 

145.  The  Unverdorben-Franchimont  reaction  with  copper 
acetate  may  be  used  as  a  special  reagent  for  resins  and  ter- 
penes. Large  pieces  of  the  parts  of  plants  to  be  examined 
may  be  placed  in  a  concentrated  aqueous  solution  of  the  salt 
named,  and  studied  after  not  less  than  about  six  days.  The 
resins  then  appear  colored  a  beautiful  emerald-green.  The 
copper  acetate  may  be  removed  before  cutting  by  washing 
with  running  water.  Pieces  so  treated  may  be  preserved  in 
50%  alcohol ;  and  microscopic  preparations  retain  their  beau- 
tiful color  in  glycerine-gelatine. 


MICRO  CHEMIS  TRY.  9 1 

Alcannin  has  also  been  much  used  (cf.  §  109)  in  the  study 
of  the  resins.  Preparations  treated  with  this  stain  do  not 
appear,  however,  according  to  the  author's  experiments,  to 
be  capable  of  preservation  in  glycerine-gelatine. 

In  connection  with  the  resins  may  be  mentioned  the 
following  compounds  not  yet  well  studied  macrochemically. 

a.   Fungus-gamboge. 

146.  Zopf  (V,  53)  denominates  as  fungus-gamboge  a 
yellow  resinous  substance  which  corresponds  in  all  its  deter- 
mined characters  with  the  gamboge-yellow  which  is  the 
chief  constituent  of  gamboge.  It  occurs  especially  in  the 
sporophore  of  Polyporus  hispidus,  chiefly  deposited  in  the 
membranes,  but  also  as  an  excretion  outside  of  the  mem- 
branes and  in  the  cell-contents.  For  its  microchemical  recog- 
nition Zopf  uses  a  solution  of  ferric  chloride^  which  colors 
fungus-gamboge,  as  well  as  membranes  containing  it,  olive- 
green  or  blackish  brown.  It  is  also  insoluble  in  water,  but 
readily  soluble  in  alcohol  and  ether ;  it  is  dissolved  with  a 
red  color  by  concentrated  nitric  or  sulphuric  acid,  and  is 
precipitated  from  these  solutions  in  yellow  flocks  on  the 
addition  of  water. 

ft.   Retinic  Acid  from    Thelephora  sp. 

147.  Zopf  has  prepared  (V,  'jj)  a  retinic  acid  from  various 
species  of  Thelephora,  which  occurs  partly  in  the  cell-con- 
tents and  is  partly  deposited  in  or  on  the  walls.  It  is  insol- 
uble in  cold  or  hot  water,  soluble  in  alcohol,  ether,  methyl 
alcohol,  petroleum-ether,  chloroform,  benzol,  carbon  bisul- 
phide, and  turpentine.  It  is  dissolved  with  a  bluish-red 
color  by  concentrated  sidpJiuric  acid,  and  thrown  down 
again  with  a  greenish-yellow  color  on  the  addition  of  much 
water. 

y.   Retinic  Acid  from    T  r  am  e  t  e  s  cinnabarina. 

148.  The  retinic  acid  prepared  by  Zopf  (V,  88)  from  the 
above-named  fungus  agrees  fully  with  the  previous  one  in 
its  behavior  with  solvents ;  but  is  distinguished  from  it  by 


92  BOTANICAL   MICROTECHNIQUE. 

being  dissolved  by  concentrated  sulphuric  acid  with  a  yellow- 
ish or  reddish  brown  color.  It  differs  from  fungus-gamboge 
in  taking  no  olive-brown  color  with  ferric  chloride. 

(5.   Retinic  Acid  from   Le7izit<is  se  pi  aria, 

149.  According  to  Bachmann  (II,  7),  opaque  globules  or 
granules  of  a  retinic  acid  are  found  on  the  membranes  of 
Lenzitcs  sepiaria  in  many  places.  These  are  quickly  dissolved 
by  a  watery  or  alcoholic  solution  of  caustic  potash  or  soda 
with  a  dark  olive-green  color. 

6.  Glucosides. 

150.  The  name  glucosides  is  given  to  a  group  of  sub- 
stances resembling  compound  ethers,  widely  distributed  in 
the  vegetable  kingdom,  which  are  characterized  by  being 
decomposed  by  various  reagents,  especially  acids,  alkalies, 
or  ferments,  into  a  species  of  sugar  and  one  or  several  other 
compounds.  Usually  this  species  of  sugar  is  glucose.  But 
there  are  commonly  included  in  the  glucosides  compounds 
which  do  not  yield  a  true  sugar,  like,  e.g.,  phloretin,  which 
forms  phloroglucin  instead  of  glucose  (cf.  Beilstein,  III,  322.) 

The  glucosides  which  yield  glucose  on  decomposition 
may  be  microchemically  recognized  by  the  aid  of  Fehling's 
solution  (cf.  §  119)  under  some  circumstances,  even  when 
they  do  not  directly  reduce  a  copper  solution,  but  only  after 
the  glucose  has  been  separated  from  them,  as  by  warming 
with  dilute  sulphuric  acid.  There  are  also  many  glucosides 
which  directly  reduce  Fehling's  solution. 

In  their  other  relations  the  glucosides  show  great  differ- 
<ences,  and  no  reactions  common  to  the  entire  group  are  yet 
known.  We  must  therefore  confine  ourselves  here  to  the 
special  description  of  some  glucosides  for  which  special  reac- 
tions, microchemically  applicable,  have  been  suggested. 

a.  Coniferin,  C.^H^Og. 

151.  Coniferin  has  been  macrochemically  produced  from 
the   cambial    juices    of   various    Coniferce,       But,    although 


MICROCHEMISTR  V.  93 

coniferin  presents  a  considerable  number  of  color-reactions, 
it  has  not  yet  been  successfully  recognized  within  the  cells 
by  microchemical  means.  Yet  the  various  reactions  for 
coniferin  succeed  easily  with  all  lignified  cell-membranes, 
and  it  is  usually  assumed  that  they  contain  coniferin 
.(cf.  §§  254-256). 

/?.  Datisci/i,  C,^H^fi^^. 

152.  Datiscin  has  been  recognized  microchemically  by  O. 
Hermann  (I,  9),  especially  in  the  cell-sap  of  the  bark-paren- 
chyma and  as  a  deposit  in  the  cell-walls  of  the  thick-walled 
cells  of  the  wood  and  bark  of  Datisca  cannabina.  This 
author  used  principally  lime  and  baryta  waters  which  give  a 
pure  yellow  solution  with  datiscin,  and  cause  a  deep  yellow 
coloring  of  all  cells  containing  it ;  while  the  addition  of 
acetic  acid  or  dilute  hydrochloric  acid  instantly  causes  the 
color  to  disappear.  Besides,  datiscin  gives  yellow  precipi- 
tates with  lead  acetate  or  zinc  chloride,  a  greenish  one  with  cu- 
pric  salts,  or  a  dark  brownish-green  one  with  ferric  chloride. 

c.  Frangulin,  C,oH,„0,o. 

153.  Frangulin  forms  a  yellow  crystalline  mass,  which  is 
insoluble  in  water,  but  dissolves  in  alkalies  with  a  cherry-red 
color.  According  to  Borscow  (I,  33),  it  occurs  inside  the 
parenchymatous  elements  of  the  stem  of  Rha^tinus  frangjila, 
united  with  small  starch-granules,  which  are  colored  blue  by 
a  solution  of  iodine,  and  bloocP-red  by  caustic  potash  or 
ammonia  (?). 

d.  Hesperidin,  C,,H„Oi,(or  C,oH,„0,,  ?). 

154-  Hesperidin  is  dissolved  in  the  cell-sap  within  the 
living  plant  ;  but  it  is  deposited,  like  inulin,  in  tissues  which 
have  lain  for  some  time  in  alcohol  or  glycerine,  in  the  form 
of  sphaerocrystals.  According  to  Pfeffer  (III),  unripe 
oranges  are  especially  favorable  objects  for  study. 

The  sphaerocrystals  of  hesperidin  are  distinguished  from 


94 


BO  TANICAL   MICRO  TECHNIQ  UE, 


those  of  inulin  by  having  usually  a  much  less  rounded  form 
than  the  latter,  and  by  showing  their  com- 
I  "-  position  from    separate  acicular  crystals 

Ilk  JLitf        much  more  clearly.     The  latter  is  the  case 

PIJF       ^Vm^   especially   in    the    smaller    spha^rites   (cf. 
^U^B  Fig.  28,  a  and  b) ;  the  larger  ones  com- 
-  ^^i^iP'      rnonly  contain  in  the  middle  a  more  ho- 

lUl:  11  mogeneous  nucleus,  as  Fig.  28,  c  shows  in 

f^  optical  median  section. 

The  hesperidin  crystals  readily  dissolve 

Fig.  28.— Hcsperidin  crys-  111.  1       . 

tais  from  alcoholic  ma- in    an    alcoholic    or  aoueous    solution   of 

terial  of  unripe  fruit  of  ,     r  •  n  n 

Citrus  A urantium.  caustU  potasu^  formmg  a  yellow  fluid,  but 
are  not  noticeably  soluble  in  cold  or  hot  water  or  in  dilute 
acids ;  while  inulin  is  at  once  completely  dissolved  by  boil- 
ing water. 

The  sphaerocrystals  of  hesperidin  are  also  soluble  in  boil- 
ing concentrated  acetic  acid,  ammonia,  and  soda  solution,  but 
are  insoluble  in  ether,  benzol,  chloroform,  carbon  bisulphide, 
and  acetone.  They  are  completely  destroyed  on  heating  to- 
a  red  heat. 

The  observation  and  preservation  of  these  crystals  in 
Canada  balsam  may  be  readily  accomplished  after  clearing 
in  oil  of  cloves.  Canada  balsam  preparations  are  especially 
adapted  to  their  study  by  polarized  light,  with  which  they 
give  the  same  appearance  as  inulin  sphaerites. 


t\   Coffec-tajinin,  C^J-i^Jd^. 

155.  Coflee-tannin,  according  to  Molisch  (I,  9),  shows  the 
following  reactions,  which  may  easily  be  followed  under  the 
microscope  on  sections  of  the  endosperm  of  the  coffee-bean. 
With  ferric  chloride  it  is  colored  dark  green,  with  ammonia 
and  caustic  potash  deep  yellow.  If  sections  are  allowed  to 
dry  with  a  drop  of  ammonia,  they  finally  take  a  green  color, 
which  at  once  changes  to  red  on  moistening  with  concen- 
trated sulphuric  acid.  An  abundant  precipitate  is  formed 
with  lead  acetate. 


MICR  O  CHE  MIS  TR  V.  95 

/.  Potassium  Myronate,  KC,oH,«NS,0.o. 

156.  Potassium  myronate,  which  occurs  in  the  seeds  of 
■many  Cruciferce^  is  spHt  up  by  the  ferment  myrosin  into  the 
allylene  mustard-oil,  glucose,  and  potassium  sulphate.  For 
its  microchemical  recognition  Guignard  (II)  first  treats  sec- 
tions with  alcohol,  which  dissolves  out  any  fatty  oil  and 
makes  the  myrosin  inactive,  while  potassium  myronate  is 
almost  insoluble  in  it.  If  the  sections  are  then  placed  in  an 
aqueous  extract  of  white  mustard-seed,  which  is  very  rich  in 
myrosin,  there  is  formed  in  the  cells  containing  myronic 
acid  the  allylene  oil  of  mustard,  which  Guignard  recognizes 
by  the  aid  of  tincture  of  Alcanna  (cf.  §  109). 

g.  Phloridzin,  Q^^^^O^^. 

157.  O.  Herrmann  (I,  21)  uses  for  the  microchemical 
recognition  of  phloridzin,  solutions  of  ferric  chloride  and 
ferrous  sulphate.  The  former  produces  a  dark  red-brown 
solution,  the  latter  a  yellow-brown  precipitate.  These 
reactions  have  led  to  trustworthy  results  only  with  Pirus 
Malus ;  in  case  of  the  pear,  cherry,  and  plum,  they  are  too 
much  masked  by  the  presence  of  large  quantities  of  tannin 
which  gives  a  green  color  with  iron  salts. 

h.  Ruberythric  Acid,  QflH^gOi^. 

158.  Ruberythric  acid  forms  the  chief  constituent  of  the 
so-called  madder  dye  in  the  roots  of  Rubia  tinctorum^  and  is 
decomposed,  on  boiling  with  hydrochloric  acid,  into  glucose 
and  alizarine  (cf.  Husemann  I,  1387).  It  has  a  yellow  color 
and,  as  Naegeli  and  Schwendener  (I,  502)  have  shown,  is 
exclusively  dissolved  in  the  cell-sap  in  young  roots,  while 
the  membranes  are  still  quite  colorless.  In  older  roots  the 
membranes  are,  however,  colored,  even  in  such  as  are  still 
living,  as  Naegeli  and  Schwendener  showed  by  plasmolysis 
of  the  cells. 

Caustic  potash  solution  colors  the  mixture  of  coloring 
matters  in  Rubia  tinctoruin  purple-red,  acids  color  it  orange, 
ferric  chloride,  orange  to  brown-red.     When  the  roots  dry, 


96  BOTANICAL   MICROTECHNIQUE. 

it  is  decomposed  with  the  formation  of  red  masses,  proba- 
bly through  the  agency  of  a  ferment. 

i.  Rutin,  C.jH^oO^- 

159.  Rutin  always  occurs  in  the  cell-contents,  according 
to  O.  Herrmann  (I,  30).  He  recommends  for  its  micro- 
chemical  recognition  ammonia  or  lime-water,  which  form 
deep-blue  solutions  with  rutin,  becoming  brown  on  expos- 
ure to  the  air. 

k.  Saffron-yellow,  Crocin,  C„H„0„. 

160.  The  crocin  contained  in  saffron  is  readily  soluble  in 
water,  less  so  in  alcohol,  and  very  slightly  so  in  ether.  It 
dissolves  in  concentrated  sulphuric  acid  with  a  blue  color 
which  soon  becomes  violet  and  finally  brown  ;  in  concen- 
trated fiiiric  acid  with,  a  deep  blue  color,  quickly  becoming 
brown  (Beilstein  HI,  357).  According  to  Molisch  (I,  57), 
the  last  two  reactions  may  well  be  used  microchemically. 

/.  Salicin,  C,^Yi,fi,. 

l6oa.  Salicin  occurs  especially  in  the  bark  of  willows  and 
poplars.  It  is  colored  a  beautiful  red  by  concentrated  sjtl- 
pkuric  acid  {RosoW  I,  8). 

m.  Saponin,  C.^H 3^0,0. 

161.  Saponin  is  dissolved  in  the  cell-sap  within  the  living 
plant,  according  to  Rosoll  (I,  11),  but  is  precipitated,  on 
drying,  in  the  form  of  amorphous  lumps  within  the  cells^ 
which  may  be  readily  observed  in  oil  or  glycerine.  They 
are  dissolved  in  water  or  very  dilute  alcohol,  but  may  be 
precipitated  from  the  solution  by  alcohol  or  ether. 

With  concentrated  sulphuric  acid  saponin  gives  at  first  a 
yellow,  then  a  bright  red,  and  finally  a  blue-violet  color.  To 
prevent  confusion  with  Raspail's  protein  reaction  (cf.  §  227), 
which  also  differs  in  the  colors  produced,  control  sections 


MICROCHEMISTRY.  9/ 

may  be  boiled  in  water  to  extract  the  saponin,  and  then 
treated  in  the  same  way  with  concentrated  sulphuric  acid. 


;/.  Solanin,  C^jH^bNOj^. 

162.  According  to  Wothtschall  (I),  who  has  tested  a  great 
number  of  reactions  as  to  their  applicability,  only  the  three 
following  are  suited  for  the  microchemical  recognition  of 
solanin. 

1.  Mandeliri  s  Reaction. — The  reagent  for  this  test  should 
be  freshly  prepared  by  dissolving  one  part  of  ammonium: 
vanadate  in  1000  parts  of  a  mixture  of  98  parts  of  concen- 
trated sulphuric  acid  with  36  parts  of  water.  This  is  added 
directly  to  the  sections  to  be  studied.  In  the  presence  of 
solanin  the  following  colors  appear  in  order  :  yellow,  orange^ 
purple-red,  brownish-red,  carmine,  raspberry-red,  violet,  blue- 
violet,  pale  greenish  blue  ;  finally  all  color  disappears.  The 
time  in  which  this  scale  of  color  is  gone  through  is  chiefly 
dependent  on  the  concentration  of  the  solutions  present, 
but  is  always  several  hours.  If  any  fatty  oils  are  present  in 
the  preparation,  which  would  also  give  color  reactions  with 
concentrated  sulphuric  acid,  they  may  be  removed  by  pre- 
liminary submersion  of  the  sections  in  ether,  in  which 
solanin  is  practically  insoluble. 

2.  Brandt's  Reaction. — .3  of  a  gram  of  sodium  selenate  is 
dissolved  in  a  mixture  of  8  ccm.  of  water  and  6  ccm.  of 
concentrated  sulphuric  acid.  After  the  addition  of  this 
reagent  to  the  sections  to  be  studied,  the  preparation  is 
gently  warmed  by  moving  it  over  a  small  flame.  As  soon 
as  the  color  appears  the  warming  must  be  stopped.  In  the 
presence  of  solanin  there  appears  first  a  raspberry-red  color, 
which  gradually  passes  into  a  currant-red,  which  soon  be- 
comes paler  and  more  brownish  yellow,  and  finally  quite 
disappears. 

3.  Sulphuric  Acid. — On  the  addition  of  concentrated  sul- 
phuric acid,  solanin  gives  at  first  a  bright  yellow  color,  which 
gradually  becomes  redder,  then  takes  a  violet  shade,  gradu- 
ally pales,  passes  into  greenish,  and  finally  quite  disappears. 


98  BOTAXICAL   MICROTECHNIQUE. 

For  the  preservation  of  plants  containing  solanin,  Wotht- 
schall  (I,  1 86)  recommends  simply  drying  them. 

0.  Syringin,  C„H„Og  +  H^O. 

163.  Syringin  occurs,  according  to  Borscow  (I,  36),  in 
branches  of  Syringa  vulgaris  within  the  thick-walled  ele- 
ments of  the  phloem  and  of  the  xylem,  as  well  as  in  the 
medullary  rays,  but  only  in  the  cell-walls.  For  its  micro- 
chemical  recognition  this  author  uses  a  mixture  of  one  part 
concentrated  sulphuric  acid  and  two  parts  water.  This  is 
added  to  delicate  longitudinal  or  transverse  sections  and 
colors  the  walls  containing  syringin  first  yellowish  green, 
after  a  few  minutes  blue,  and  finally  violet-red. 

p.  Glucoside  (?)  from  the  Sti7nulus-conducting  Tissue  of 
Mimosa  pudiea. 

164.  Haberlandt  (I,  17)  has  observed  that  when  the  stem 
•dr  petiole  of  Mimosa  pudiea  is  cut,  the  drops  of  fluid  which 
escape  leave,  on  drying,  a  considerable  quantity  of  a  crys- 
tallized substance  whose  composition  has  not  yet  been  made 
out,  but  which  should  be  for  the  present  included  among 
the  glucosides,  on  account  of  its  reactions. 

The  crystals  of  this  substance  are  always  colored  pale 
brownish  and  show  very  various  forms.  They  sometimes 
form  large  prisms  or  cross-shaped  twins,  sometimes  gland- 
like masses  or  dendritic  bodies ;  and  sphaeritic  formations 
have  been  seen.  The  material  identity  of  these  different 
formations  is  shown  by  the  fact  that  they  all  dissolve  with  a 
red-violet  color  in  ferric  chloride.  They  are  also  soluble  in 
water,  but  very  slightly  so  in  alcohol,  and  quite  or  almost 
quite  insoluble  in  ether.  They  are  dissolved  by  concen- 
trated sulphtiric  acid  with  a  yellow-green  color,  which  be- 
comes red-brown  on  warming.  Dilute  sulphuric  and  hydro- 
chloric acids  throw  down  from  the  aqueous  solution  a  finely 
granular  white  precipitate,  which  is  soluble  in  alcohol. 
Ferrous  sulphate  causes  a  deep  rust-red  color.  Lead-acetate 
produces  a  heavy  yellow  precipitate  in  the  aqueous  solution, 


MICR  O  C HEM  IS  TR  Y.  99 

which  is  soluble  in  acetic  acid.  The  substance  does  not 
directly  reduce  Fehling's  solution,  but  does  so  after  heating 
with  dilute  sulphuric  acid. 

Since  the  isolation  of  this  questionable  substance  has  not 
yet  been  accomplished,  it  must  remain  doubtful,  in  case  of 
several  of  the  reactions  described,  how  far  they  are  influenced 
by  other  substances  also  contained  in  the  drops  of  fluid. 

7.  Bitter  Principles  and  Indifferent  Substances. 

a.   Calycin,  CigHj^O^. 

165.  Calycin  occurs  in  the  form  of  small  yellow  crystals 
deposited  on  the  membranes  of  Calyciiim  chrysocephalunt 
and  various  other  lichens.  For  its  microchemical  recog- 
nition it  is  best,  according  to  Bachmann,  to  treat  with  gla- 
cial acetic  acid  a  bit  of  the  lichen  under  examination,  which 
has  been  rubbed  as  fine  as  possible,  then  to  bring  the  whole 
together  into  a  drop  and  let  it  evaporate.  The  calycin  crys- 
tallizes out  in  long,  acicular,  strongly  doubly  refractive 
crystals. 

It  is  also  characteristic  of  calycin  that  it  is  not  dissolved 
by  caustic  potash  solution  and  suffers  no  change  of  color 
by  it. 

b,  Spergulin,  (C,H,OJx. 

166.  Harz  (II)  has  isolated  from  the  seed-coats  of  Sper- 
gula  vulgaris  and  5.  maxima  a  strongly  fluorescent  sub- 
stance which  he  calls  spergulin.  It  is  readily  soluble  in 
absolute  and  dilute  alcohol  and  appears  colorless  or  faintly 
greenish  or  olive-brown  in  this  solution,  showing  an  in- 
tense, dark-blue  fluorescence,  while  a  beautiful  emerald- 
green  fluorescence  appears  after  the  addition  of  a  small 
quantity  of  alkali.  It  cannot  be  obtained  in  crystalline 
form  from  this  solution. 

It  is  also  characteristic  of  spergulin  that  it  dissolves  in 
concentrated  sulphuric  acid  with  a  beautiful  dark  blue  color. 
It  is  readily  soluble  in  methyl  alcohol,  less  so  in  amyl  alco- 
hol, and  hardly  so  in  petroleum  and  ether. 


lOO  BOTANICAL   M ICROTECHXIQUK. 

It  is  insoluble  in  fatty  and  ethereal  oils,  in  benzine,  carbon 
bisulphide,  chloroform,  phenol,  cold  and  hot  water,  and 
dilute  organic  and  inorganic  acids. 

Since  the  membranes  of  the  strongly  thickened  outermost 
cell-layer  of  the  seed-coat  dissolve  in  concentrated  sulphuric 
acid  with  a  deep-blue  color,  Harz  concludes  that  spergulin 
is  contained  in  the  membranes  themselves. 

8.  Coloring  Matters. 

167.  The  compounds  united  under  this  head  do  not  form 
a  group  of  chemically  related  substances,  but  there  are  in- 
cluded here  all  colored  substances  whose  chemical  constitu- 
tion is  still  too  little  known  to  enable  them  to  be  placed  in 
their  proper  position  in  the  natural  series.  Concerning 
most  of  them,  then,  our  knowledge  is  extremely  incomplete, 
and  an  actual  isolation  and  quantitative  analysis  has  been 
carried  out  with  any  exactness  upon  very  few  of  them. 

It  cannot  be  my  duty  to  enumerate  all  the  coloring  mat- 
ters heretofore  extracted  from  various  parts  of  plants.  It 
seems  rather  that  I  should  restrict  myself  to  those  concern- 
ing whose  chemical  relations  we  have  some  trustworthy 
information,  and  which,  especially,  are  microchemically 
recognizable  with  some  certainty. 

I  have  grouped  the  coloring  matters  according  to  the 
place  in  the  living  plant  where  they  occur. 

a.    The  Pigments  of  the  ChromatopJiorcs. 

168.  The  different  colorings  of  the  chromatophores,  ac- 
cording to  our  present  knowledge,  if  we  omit  for  the 
moment  the  algae  which  are  not  green,  are  to  be  referred  to 
a  relatively  small  number  of  coloring  matters.  But  these 
arc  still  too  little  studied  to  make  a  completely  certain 
limitation  possible.  Four  coloring  matters  may  be  distin- 
guished with  some  certainty,  and  I  will  here  confine  my- 
self to  their  brief  description.  A  special  discussion  of  the 
very  abundant  literature  of  chlorophyll  does  not  seem  de- 
sirable here,  since  it  contains  almost  exclusively  the  results 


MICR  O  CHE  MIS  TR  V.  I O  r 

of  macroscopic   investigations   and    has  hitherto   produced 
very  few  results  of  physiological  value. 

After  the  discussion  of  these  four  pigments,  the  coloring- 
matters  of  the  chromatophores  of  the  algae  which  are  not 
green  will  be  taken  up. 

(X.   Chlorophyll-green. 

169.  The  chloroplasts  or  chlorophyll-grains  seem  to  owe 
their  color  in  all  cases  to  one  and  the  same  coloring  matter, 
to  which  is  commonly  given  the  name  chlorophyll-green,  or 
chlorophyll.  It  is  macrochemically  distinguished  by  its 
strong  red  fluorescence  and  by  its  absorption  spectrum,  in 
which  may  be  distinguished  four  bands,  besides  the  end 
absorption  beginning  in  the  blue.  Of  these  the  strong  band 
in  the  red  is  especially  characteristic.  Since  no  other  green 
coloring  matter  has  yet  been  observed  in  the  chromatophores, 
it  is  only  in  case  of  feebly  colored  chloroplasts  that  any 
doubt  can  arise  as  to  the  presence  of  chlorophyll  in  micro-^ 
scopical  observation.  In  such  cases  the  so-called  Jiypochlorin 
or  cJiloropJiyllan  reaction  has  been  used  for  the  recognition 
of  chlorophyll-green.  According  to  A.  Meyer  (II),  this 
reaction  is  best  conducted  by  treating  the  sections  under  a 
cover-glass  with  glacial  acetic  acid.  There  are  then  ex- 
truded on  the  surfaces  of  the  chromatophores,  sometimes 
crystalline,  sometimes  amorphous  masses  which  contain 
decomposition-products  of  chlorophyll.  For  further  reac- 
tions of  hypochlorin,  see  A.  Meyer,  II. 

ft.  Carotin,   Chlorophyll-yellow. 

170.  Carotin  was  first  prepared  from  the  roots  of  Daiicus 
Carota,  where  it  occurs  in  the  form  of  rhombic  plates  or 
variously  shaped  crystalline  formations  (cf.  §  356).  The 
same  substance  also  occurs  in  the  orange  or  red  chromat- 
ophores of  many  flowers  and  fruits.  But,  according  to  the 
researches  of  Arnaud  (II),  it  is  constantly  to  be  met  with 
within  the  chloroplasts,  which,  according  to  Immendorff  (I)^ 
contain  no  yellow  or  yellowish-red  coloring  matter  except 
carotin.     Carotin  would  thus  be  identical  with  the  coloring; 


102  BOTANICAL   MICROTECHNIQUE. 

matters  called  chlorophyll-yellow,  xanthophyll,  erythrophyll, 
chrysophyll,  etc.  According  to  Immendorff  (I,  516),  carotin 
also  occurs  in  the  chromatophores  of  etiolated  parts  of 
plants  and  is  the  cause  of  the  autumnal  yellowing  of  leaves. 

171.  The  elementary  composition  of  carotin  corresponds 
to  the  formula  C„H,3,  according  to  Arnaud  (I)  ;  it  would 
therefore  clearly  constitute  an  hydrocarbon.  According  to 
Immendorff  (I,  510),  it  is  precipitated  in  shining,  deep-red 
crystals  by  alcohol,  from  its  solution  in  carbon  bisulphide, 
which  it  colors  deep  blood-red.  These  crystals  show,  when 
undecomposed,  a  strong  dichroism  (cf.  §  356,  note)  ;  but  on 
lying  in  the  air  they  take  up  oxygen  and  pass  over  gradually 
into  a  colorless  compound  readily  soluble  in  alcohol.  On 
warming  they  first  take  a  brick-red  color,  and  above  160°  C. 
they  melt. 

Courchet  has  prepared  very  variously  shaped  crystals  of 
carotin  from  various  parts  of  plants  which  showed,  like 
those  observed  naturally,  sometimes  a  red,  sometimes  a 
more  yellow  color.  It  is  still  unknown  to  what  these  differ- 
ences in  color,  which  are  to  be  seen  in  one  and  the  same 
cell  in  the  carrot,  are  to  be  referred.  It  is  also  yet  to  be 
determined  whether  all  the  coloring  matters  called  carotin 
are  identical ;  for  it  seems  probable  that  we  have  to  do  here 
at  least  with  a  group  of  nearly  related  coloring  matters. 

172.  The  following  reactions  may  serve  for  the  micro- 
chemical  recognition  of  carotin  :  With  a  solution  of  iodine 
{e.g.,  aqueous  solution  of  iodine  and  iodide  of  potassium),  it 
is  colored  greenish  or  greenish  blue ;  with  concentrated  suL 
phtiric  acid,  first  violet  and  then  indigo-blue  ;  from  its  deep 
blue  solution  in  concentrated  sulphuric  acid  it  is  precipitated 
in  green  non-crystalline  flakes,  on  the  addition  of  water,  ac- 
cording to  Immendorff  (I,  509). 

Carotin  is  also,  according  to  Arnaud  (I),  insoluble  in 
water,  almost  so  in  alcohol,  very  slightly  soluble  in  ether, 
more  readily  so  in  benzine,  and  most  so  in  chlorofonn  and 
carbon  bisulphide. 

These  solutions,  even  when  from  carmine-red  crystals,  are 
colored  yellow  or  orange-yellow,  according  to  their  concen- 


MICRO  CHE  MIS  TR  V.  1 0$ 

tration,  while  the  solution  of   carotin  in    carbon   bisulphide, 
as  already  stated,  is  always  blood-red. 

The  absorption  spectrum  of  carotin  shows,  according  to 
Immendorff  (I,  510),  two  bands  in  the  blue  and  absorption 
of  the  violet. 

y.  Xa  n  t  h  i  n. 

173.  Xanthin  occurs  in  the  yellow  chromoplasts,  always- 
in  amorphous  form,  and  especially  in  small  granules  (grana)- 
(cf.  §  357).  Its  alcoholic  solution  leaves,  on  evaporation, 
according  to  Courchet  (I,  349),  a  wholly  amorphous,  resin-^ 
like  mass.  It  is  insoluble  in  water,  little  soluble  in  ether, 
chloroform,  and  benzine,  but  more  so  in  alcoJiol.  With  con- 
centrated sulphuric  acid,  the  isolated  pigment,  as  well  as  the 
chromoplast,  takes  first  a  greenish,  then  a  blue  color ;  with 
iodine,  best  used  in  the  form  of  the  solution  with  potassium 
iodide,  it  becomes  green. 

(5.  Coloring    Matter    of    Aloe    Flowers. 

174.  A  coloring  matter  whose  reactions  differ  essentiall)^ 
from  those  of  xanthin  and  carotin  has  been  recognized  by 
Courchet  (I)  in  the  chromoplasts  of  the  flowers  of  Aloe. 
It  is  insoluble  in  ether  and  chloroform,  but  readily  so  in 
alcohol^  with  a  currant-red  color.  On  dilution  its  solution 
becomes  rose-red ;  benzine  takes  up  but  little  of  it  ;  and  it 
has  not  yet  been  obtained  in  crystalline  form.  It  becomes 
yellowish  green  with  sulphuric  acid,  and  about  the  same  with. 
hydrochloric  acid ;  nitric  acid  first  turns  it  yellow  and  then 
decomposes  it.  Caustic  potasJi  colors  the  granules  of  the 
coloring  matter  orange  and  makes  them  run  together ; 
iodine  colors  them  dirty  yellow. 

This  substance  has  not  yet  been  observed  in  other  plants. 

6.  Coloring    Matters   of   the   Chromatophores   of    the 
Flor  idets  . 

175.  In  the  chromatophores  of  the  Floridece  there  occur  a 
red  pigment  soluble  in  water  and  a  green  pigment  soluble 
in   alcohol.      The    latter  is   almost  certainly  identical  with 


104  BOTANICAL   MICROTECHNIQUE. 

chlorophyll.     The  pigment  soluble  in  water   is   at   present 
commonly  called  phycoerythrin. 

Phycoerythrin  is,  according  to  Schutt  (II  and  IV),  in- 
soluble in  alcohol,  ether,  benzole,  benzine,  carbon  bisul- 
phide, glacial  acetic  acid,  and  fatty  oils.  Its  saturated 
aqueous  solution  appears  by  transmitted  light  dark  bluish 
red,  by  reflected  light  more  or  less  yellow,  on  account  of  its 
deep  orange-yellow  fluorescence.  Its  absorption-spectrum 
is  distinguished  from  that  of  chlorophyll,  according  to 
Schutt's  investigations  (II),  especially  by  the  fact. that  its 
maximum  of  brilliancy  is  at  the  point  in  the  red  where  the 
strongest  absorption-band  of  chlorophyll  occurs, 

C.  Coloring    Matters    of   the    P  hceop  hy  ce  ce .. 

176.  Millardet  has  recognized  three  pigments  in  the  chro- 
matophores  of  the  Phceophycece :  chlorophyllin,  phycoxan- 
thin,  and  phycophaein.  The  first  two  of  these  are,  accord- 
ing to  Hansen  (II),  identical  with  chlorophyll-green  and 
chlorophyll-yellow ;  that  is,  with  chlorophyll  and  carotin. 

Phycophcem  is  readily  soluble  in  water,  especially  in  hot 
water;  its  saturated  solution  has  a  deep  red-brown  color 
and  shows  with  the  microspectroscope  a  regular  increase  of 
absorption  from  the  red  to  the  blue  end  of  the  spectrum 
without  any  absorption-bands.  It  is  also  little  soluble  in 
dilute,  and  insoluble  in  absolute,  alcohol,  as  also  in  ether, 
carbon  bisulphide,  benzol,  benzine,  and  fatty  oils.  It  is 
more  or  less  completely  precipitated  by  acids  from  its 
aqueous  solution,  incompletely  so  by  caustic  soda,  and  not 
at  all  by  ammonia  and  salts  of  the  alkalies.  Salts  of  the 
alkaline  earths  precipitate  it  (Schutt  III). 

t}.  Coloring   Matters    of   the    Cy  anop  hy  c  e  a . 

177-  Three  different  pigments  have  been  isolated  by 
Reinke  (II,  405)  from  an  Oscillatoria,  which  he  calls  chloro- 
phyll, phycoxanthin,  and  phycocyanin.  Of  these  the  first 
must  certainly  be  identical  with  ordinary  chlorophyll,  but 
whether  the  phycoxanthin  is  to  be  identified  with  carotin  is 


MICROCHEMISTKY.  IO5 

not   yet   certain,   since,   according   to    Reinke's  statements, 
this  is  not  very  probable. 

PJiycocyanin  is  soluble  in  water,  and  in  this  solution  has  a 
bright  blue  color  with  a  red  fluorescence.  Its  absorption- 
spectrum  shows  four  bands,  according  to  Reinke. 

O.   Coloring    Matters    of    the    Diatom  a  cetv. 

178.  The  chromatophores  of  the  DiatomacecE,  which  are 
colored  yellowish  brown  in  the  living  state,  are  colored 
greenish  by  alkalies  and  dilute  acids,  and  by  concentrated 
sulphuric  acid  a  beautiful  verdigris-green,  as  Naegeli  has 
shown.  They  contain  at  least  two  pigments,  one  of  which 
is  surely  identical  with  chlorophyll,  while  the  other  is  often 
called  phycoxanthin,  but  more  appropriately  diatomin. 

Diatomin  is  characterized  by  being  soluble  in  dilute 
alcohol  with  a  brownish-yellow  color.  It  is  not  probable, 
from  the  investigations  already  made,  that  it  is  identical 
with  carotin  (cf.  Askenasy  I,  236  and  Nebelung  I,  394). 

I.    Coloring    Matters    of   the    Peridinex. 

179.  The  PeridinecB,  which  have  recently  been  commonly 
included  among  the  Algae,  are  distinguished,  as  Klebs  (V, 
732)  has  shown,  by  the  possession  of  true  chromatophores. 
These  contain,  according  to  Schiitt  (I),  three  pigments, 
which  he  calls  Peridine-chlorophyllin,  peridinin,  and  phyco- 
pyrrin.  Of  these,  Peridine-chlorophyllin  is  either  identical 
with,  or  very  closely  related  to,  chlorophyll. 

1.  Peridinin  is  insoluble  in  water,  but  readily  dissolves  in 
nlcoholy  giving  a  fluid  of  the  color  of  red  wine.  It  is  also 
readily  soluble  in  ether,  chloroform,  benzol,  carbon  bi- 
sulphide, and  glacial  acetic  acid.  Its  absorption-spectrum 
shows,  according  to  Schutt  (I),  the  Band  I  of  chlorophyll 
between  B  and  C,  and  is  also  marked  by  a  strong  absorp- 
tion in  the  green-yellow. 

2.  Phycopyrrin  (from  nvppo^y  red-brown)  is  soluble  iny 
water,  giving  a  dark  brown-red  fluid.  It  is  also  easily 
soluble  in  alcohol,  ether,  bisulphide  of  carbon,  and  benzol. 


Io6  BOTANICAL   MICROTECHNIQUE. 

The  absorption-spectrum  of  the  aqueous  solution  has,  ac- 
cording to  Schiitt,  a  certain  similarity  to  the  chlorophyll 
spectrum,  in  that  it  shows  Band  I. 

b.  Fatty  Pigments  or  Lipochromes. 

180.  At  present,  all  those  yellow  or  red  pigments  which 
are  colored  blue  by  sulphuric  or  'nitric  acid,  and  green  by 
solutions  of  iodine  with  potassium  iodide,  are  called  fatty 
pigments  or  lipochromes.  They  are  mostly  dissolved  in 
fatty  substances  within  the  living  cell. 

Zopf  (IV)  has  recently  investigated  the  relations  of  the 
lipochromes,  especially  toward  sulphuric  acid,  and  has 
proved  that,  in  this  reaction,  deep-blue  crystals  often  occur, 
which  he  calls  lipocyanin  crystals.  These  are  formed  espe- 
cially when  rather  dilute  sulphuric  acid  is  added  to  the 
residue  from  an  evaporated  solution  of  a  lipochrome  upon 
the  slide.  Zopf  also  succeeded  in  obtaining  lipocyanin 
crystals  directly  from  various  organs  by  letting  the  sections 
dry  before  treating  them  with  sulphuric  acid. 

181.  According  to  their  chemical  relations,  the  already 
described  pigments,  carotin  and  xanthin,  belong  to  the  lipo- 
chromes (cf.  §§  170  and  173).  There  also  belongs  here  the 
red  pigment  prepared  by  Cohn  from  the  cells  of  Hcejnato- 
coccus,  which  he  calls  Juvmatochrome.  The  pigment  called 
chlororufin  by  Rostafinski  (I)  is  also  to  be  included  here. 
Zopf  and  Bachmann  have  prepared  various  lipochromes 
from  different  fungi  (cf.  Zopf  II,  414).  Finally,  the  bacterio- 
purpurin  prepared  by  Lankaster  from  various  red  Bacteria 
is  to  be  placed  among  the  lipochromes,  according  to  Biit- 
schli  (I,  9),  whose  studies  indicate  that  it  is  identical  with 
Cohn's  haematochrome. 

Whether  all  these  pigments  are  nearly  related  chemically, 
and  how  far  they  are  identical  with  each  other,  or,  on  the 
contrary,  consist  of  groups  of  more  or  less  different  pig- 
ments, must  be  determined  by  further  investigations. 


MICR  O  CHEMIS  TRY.  I O/ 

c.  Other  Coloring  Matters  dissolved  in  Fats  or  Ethereal  Oils. 

182.  Of  the  other  pigments  which  from  their  chemical 
reactions  do  not  belong  to  the  Hpochromes,  but  occur  dis- 
solved in  fats  or  ethereal  oils  in  the  living  plant,  only  curcu- 
min  has  been  microchemically  investigated. 

Curcumin,  C14H44O4. 

Curcumin  occurs,  according  to  O.  Herrmann's  investiga- 
tions (I,  24),  in  the  fresh  rhizome  of  Curenma  ainata  within 
the  parenchymatous  cells  of  the  fundamental  tissue,  dis- 
solved in  an  ethereal  oil.  This  author  uses,  for  its  micro- 
chemical  recognition,  lead  aeetate,  which  forms  a  brick-red 
precipitate  with  the  curcumin,  and  sulphnric  aeid,  which 
colors  it  crimson. 

d.   Coloring  Matters  dissolved  in  the  Cell-sap. 

183.  The  pigments  dissolved  in  the  cell-sap  have  been 
little  studied.  There  are  usually  distinguished  only  two 
different  pigments  or  groups  of  closely-related  compounds  : 
namely,  anthocyanin  or  cyanin,  and  anthochlorin  or  xanthein. 
The  first  of  these  produces  red,  blue,  or  blue-green  colors, 
and  the  latter  the  yellow  and  yellow-brown  tones. 

a.   Anthocyanin. 

184.  Anthocyanin  is  readily  soluble  in  water,  and  has  iiT 
this  solution,  according  as  its  reaction  is  more  or  less  acid 
or  alkaline,  a  red,  violet,  blue,  blue-green,  green,  or  yellow^ 
green  color.  It  is  wholly  decolorized  by  strong  alkalies.  It 
is  also  soluble  in  alcohol  and  ether. 

Whether  all  the  pigments  called  anthocyanin  are  chemi- 
cally identical  must  be  determined  by  future  studies.  Han- 
sen (III)  believes  in  the  identity  of  the  red  and  blue  pig- 
ments prepared  from  various  organs,  on  the  ground  of  his 
spectroscopic  investigations. 

But  N.  J.  C.  Miiller  (I)  has  recently  endeavored  to  show 
that  a  considerable  number  of  very  different  compounds 
have    heretofore    been    called    anthocyanin.       But,    as   this 


I08  BO  TANICAJ^  MICRO  TECHNIQ UE. 

author  gives  no  account  of  the  mode  of  preparation  of  his 
solutions,  which  were  studied  only  as  to  their  behavior  with 
caustic  potash  and  sulphuric  acid,  and  with  the  spectroscope, 
it  remains  uncertain  how  far  their  different  behavior  is  to  be 
attributed  to  foreign  admixtures. 

I  may  remark  here  that  secretions  of  pigment  of  a  blue 

or  violet  color  and  of  a  granular 
or  crystalline  structure  have 
been  observed  in  various  plants. 
These  consist  most  probably  of 
a  compound  of  anthocyanin 
with  another  substance  not  yet 
recognized,  perhaps  a  tannin. 
F.O.  „.-rei.  of  iheepidermis  of  a  petal       Thcsc  sccrctions  may  be  finely 

oiDelphiniu,n/or,nosu,n.  ^^^^      -^^      ^J^^      epldcrmis     of     thc 

petals  of  various  species  of  Delphinium.  In  the  cell  from 
the  epidermis  of  the  petal  of  D.  formosiun,  shown  in  Fig. 
29,  the  beautifully  blue  secretions,  which  consist  plainly  of 
delicate  needles,  lie  in  the  violet  cell-sap.  But  in  this  place 
the  pigment  deposits  may  present  the  most  various  forms. 

fi.    Anthochlorin. 

185.  The  yellow  pigments  dissolved  in  the  cell-sap  are 
distinguished  from  xanthin,  which  occurs  in  the  chromato- 
phores,  by  never  being  colored  blue  by  concentrated  sul- 
phuric acid,  according  to  Courchet  (I,  361-2).  In  other 
respects,  the  yellow  pigments  contained  in  the  cell-sap  of 
various  plants  show  very  different  relations  to  chemical 
reagents,  as  Courchet  has  shown.  But  our  knowledge  of 
Ihem  is  still  too  fragmentary  to  permit  their  classification. 

€.  Coloring  Matters  ivhich  are  first  contained  in  thc  Cell-con- 
tents, but  later  penetrate  the  Wall. 

186.  According  to  Sanio  (II,  202),  Naegeli  and  Schwen- 
dener  (I,  501),  and  Pracl  (I,  67),  all  or  nearly  all  the  pig- 
ments of  the  dye-woods  belong  in  this  category.  This 
follows  with  greater  probability  from   the  study  of   dried 


MICK  0  C HEM  IS  TR  Y.  I O9 

Avoods,  which  usually  contain  in  the  cavities  of  the  cells  of 
the  medullary  rays  and  of  the  wood-parenchyma,  granules 
of  the  same  character  as  the  pigments  which  incrust  their 
walls. 

Naegeli  and  Schwendener  have  studied  from  this  point  of 
view  the  madder  pigments  and  berberin,  which  have,  how- 
ever, recently  been  placed  in  other  parts  of  the  system  of 
organic  compounds  (cf.  §§  158  and  212). 

187.  According  to  the  investigations  of  A.  Rosoll  (I,  137), 
the  yellow  pigment  contained  in  the  bracts  of  the  involucre 
of  various  species  of  Helichrysum,  which  he  calls  iLelichrysiUy 
belongs  to  this  group.  According  to  Rosoll,  it  is  associated 
with  the  protoplasm  in  the  young  bracts,  and  only  pene- 
trates the  membrane  in  old  cells.  Helichrysin  has  not  yet 
been  studied  macrochemically,  but  it  is  characterized  micro- 
chemically  by  being  soluble  with  difficulty  in  cold  water, 
but  readily  so  in  hot  water,  alcohol,  ether,  and  organic 
acids.  It  is  insoluble  in  benzol,  chloroform,  and  carbon 
bisulphide.  Mineral  acids,  as  well  as  alkalies,  color  it  a 
beautiful  purple-red.  Lead  acetate  precipitates  the  pigment 
from  its  solution  with  a  red  color. 

188.  It  seems  also  probable,  from  the  researches  of  Naegeli 
and  Schwendener  (I,  504),  that  anthocyanin  can  incrust  the 
cell-walls  under  some  circumstances.  At  least,  the  pigment 
extracted  from  the  seed-coat  of  Abriis precatoriics  showed  a 
relation  to  acids  and  alkalies  quite  corresponding  to  that  of 
anthocyanin  ;  while,  on  the  other  hand,  membranes  com- 
pletely washed  out  were  deeply  stained  by  the  cell-sap 
pressed  from  red  petals  of  flowers. 

/.   Coloring  Matters  which  only  occur  deposited  in  the  Cell- 
wall. 

189.  Coloring  matters  with  the  above  characteristic  are 
widely  distributed  among  the  lower  plants.  Thus  two  pig- 
ments are  found  among  the  Cyanophycece,  according  to  Nae- 
geli and  Schwendener  (I,  505),  gloeocapsin  and  scytonemin, 
which  are  completely  restricted  in  their  occurrence  to  the 


I  lO  BOTANICAL   MICR07i:CHNIQUE, 

cell-wall,  and  never  occur  in  the  cell-contents ;  so  that  it 
must  be  supposed  that  they  are  formed  directly  in  the  wall. 
Glccocnpsin  is  red  or  blue,  and  becomes  red  with  hydro- 
chloric acid  (rose-red,  reddish  orange,  or  bluish  red),  and  blue 
or  blue-violet  \v\X.\\  caustic  potash.     It  occurs  chiefly  in  Gla'o- 

capsa, 

Scytoncmin  gives  a  yellow  to  dark-brown  color  to  the 
membranes  of  Scytotiema  and  various  other  algae.  We  have 
recent  studies  of  its  characteristics  by  Correns  (I,  30). 
According  to  these,  it  is  especially  distinguished  by  taking 
a  blue-violet  color,  greatly  resembling  that  which  appears  in 
the  cellulose  reaction,  with  chloroiodide  of  zinc,  or  with  iodine 
and  sulphuric  acid.  Scytonemin  is  destroyed  by  can  de 
Javelle  (cf.  §  12,  4),  and  threads  so  treated  no  longer  give 
the  above-mentioned  reactions  with  iodine. 

With  acids  scytonemin  takes  a  green  color  which  is 
restored  to  the  original  color  on  the  neutralization  of  the 
acid.  Even  sulphurous  acid  acts  in  this  way  and  does  not 
destroy  the  pigment. 

Alkalies  color  it  more  red-brown.  Sheaths  which  had 
been  treated  with  caustic  potash,  although  the  pigment 
appeared  unchanged  after  the  potash  was  washed  out,  no 
longer  took  the  violet  color  with  chloroiodide  of  zinc,  accord- 
ing to -Correns.  No  macrochemical  studies  of  the  compo- 
sition of  these  substances  have  been  made. 

190.  Very  numerous  and  various  pigments  occur  in  the 
membranes  of  the  fungi,  according  to  the  investigations  of 
Bachmann  (II)  and  Zopf  (V).  But,  since  these  have  hitherto 
been  studied  almost  exclusively  in  their  macroscopic  rela- 
tions, and  very  little  has  been  established  as  to  their  chemi- 
cal composition,  the  student  may  be  referred  to  Zopf's 
account  of  them  (II,  418). 

191.  The  pigments  deposited  in  the  membranes  of  the 
lichens  have  lately  been  studied  by  Bachmann  (IV).  He 
distinguishes  by  their  microchemical  behavior  sixteen  differ- 
ent pigments,  whose  chief  reactions  are  shown  upon  the 
table  on  the  next  page. 


MICROCHEMISTR  V. 


Ill 


1 

n 

.2 

I 

10 

first  KOH,  then  HChblue 
HNO3  :  brighter  and  purer  green 
HCl:  violet 
HCl :  indistinct  purple-red 

HjO.  insoluble  3 

first    KOH,   then    HNO,,  then 
H2SO4  :  violet  crystals 

first   KOH.   then    H0SO4,  then 
HNO3  :  blackish 

dilute  H2SO4  :  bright  yellow 

cone.  H,S04  :  deep  violet,  finally 
gray 

CaClaOa  :  first  blue,  then  gray  ; 
finally  decolorized 

d 

a: 

III 

a               "^ 

d 

«             g.       -i        ^   -s                       >          ^'    1 

s-s     1  1  ^  £ii  :  s                .       g^^ 

r   '^  -1 1  s'p  It          €    "1  s 

1                     c      0     30-0      0     .^                                     •=                 ^«   -So 

o 

oa 

B     rt  w   0 

E 

E 

o 

liiilf'  iii.h    ii 

0     ?                     «             ^ 

"o  *J 

u 

iMMt«be.2         -^          u|3        SS-5        oc         ^-£3 

1          "  ^   -    ^  1  1    1.  1     1  1 

s 

si 

1  ,i  ^  '  s 

mil  E  i    i  it|!ii-i|i  1  i 
•Mill   1   1     ||.-liiir|i|  i 

112  BOTANICAL   MICKOTECHNIQUE. 

g.  Coloring  Matters  which  are  deposited  upon  the  Ccll-ivall. 

192.  In  many  lichens  colored  compounds  occur  which  are 
attached  externally  to  the  membranes.  They  are  mostly 
crystalline,  more  rarely  amorphous. 

Amorphous  excretions  of  pigment  were  found  by  Bachmann 
(IV,  27)  only  in  two  lichens,  and  he  has  named  them,  from 
the  lichens  in  which  they  occur,  Arthonia-violet  and  Urceo- 
laria-red.  Arthonia-violet  occurs  in  all  parts  of  Arthonia 
gregaria  and  is  especially  distinguished  by  being  somewhat 
soluble  in  cold,  but  readily  so  in  hot,  water.  It  is  also  solu- 
ble in  alcohol  with  a  wine-red  color.  It  is  dissolved  by  a 
solution  of  caustic  potash  with  a  violet  color,  but  is  insoluble 
in  lime  and  baryta  waters.  It  dissolves  in  sulphuric  acid 
with  an  indigo-blue  color,  passing  later  into  mallow-red ; 
and  in  nitric  acid  with  a  red  color. 

Urceolaria-red  occurs  in  the  thallus  of  Urceolaria  ocellata. 
It  is  characterized  microchemically  by  not  being  changed  by 
alcohol,  lime-water,  or  ammonium  carbonate.  It  is  dissolved 
with  a  greenish-brown  color  by  caustic  potash  solution  and 
baryta-water,  as  well  as  by  concentrated  nitric  and  sulphuric 
acids.     A  solution  of  calcium  chloride  decolorizes  it. 

193.  The  substances  already  described  elsewhere,  emodin,. 
chrysophanic  acid,  and  calycin  (cf.  §§  140,  141,  and  165),. 
belong  to  the  crystalline  excretions  of  the  lichen-fungi,  as- 
also  a  series  of  other  so-called  lichen  acids,  which  have  here- 
tofore been  studied  almost  exclusively  macroscopically  (cf. 
Schwarz  II  and  Zopf  II,  401). 

But  I  will  discuss  somewhat  more  in  detail  certain  recently 
described  fungus-pigments. 

ix.  Thelephoric  Acid. 

194.  Zopf  (V,  81)  designates  as  thelephoric  acid  a  pigment 
extracted  from  various  species  of  Thelephora,  whose  solu- 
tions are  of  a  beautiful  red  color,  while  the  solid  crystalline 
pigment  has  a  violet-blue  or  indigo-blue  color.  This  partly 
forms  an  incrustation  of  the  membrane,  partly  a  crystalline 
deposit  upon  it. 


MICR  O  CHEMIS  TK  Y.  I  I  3 

Its  behavior  with  aniniojiia,  which  gives  it  a  splendid  blue 
color,  is  especially  characteristic  of  thelephoric  acid  ;  while 
caustic  potash  and  soda  produce  a  more  bluish-green  color. 
Thelephoric  acid  is  also,  according  to  Zopf,  insoluble  in 
water,  ether,  chloroform,  petroleum-ether,  carbon  bisul- 
phide, and  benzol,  but  is  pretty  readily  dissolved  by  warn-^ 
alcoJiol.  Concentrated  sulphuric  or  hydrochloric  acid  neither 
changes  the  color  nor  dissolves  the  solid  pigment ;  but  con- 
centrated acetic  <^^/^  dissolves  it  with  a  rose-red  or  wine-red 
color,  nitric  acid  with  a  yellow  color,  and  dilute  chromic  acid 
with  a  dark  chrome-yellow  color. 

ft.   Xanthotrametin. 

195.  Zopf  calls  by  the  name  xanthotrametin  a  red  pig- 
ment which  is  deposited  on  the  membranes  of  Tranietes 
cinnabarina  in  the  form  of  granular  brick-red  incrustations. 
This  dissolves  in  concentrated  nitric  acid  with  a  deep  orange- 
red  color,  in  JiydrocJdoric  acid  with  an  orange-yellow,  and 
then  more  reddish,  shade,  in  srdpJmric  acid  with  a  rose-red 
color,  and  in  glacial  acetic  acid  with  a  yellow  color.  Dilute 
sulphuric  acid  dissolves  it  with  an  at  first  orange-yellow 
color  which  then  becomes  redjder. 

Xanthotrametin  is  dissolved  by  ammonia  and  sodium  car- 
bonate with  a  yellow  color,  by  dilute  caustic  soda  and  lime- 
water  with  a  yellow  color,  becoming  paler,  and  by  dilute 
caustic  potash  with  a  yellow  color  which  quickly  passes  into 
reddish.     In  ferric  chloride  it  is  insoluble. 

y .   Pigment    of    A  g  ar  i  c  u  s  a  r  m  i  1 1  a  t  u  s  . 

196.  This  forms,  according  to  Bachmann  (II,  7),  crystal- 
line cinnabar-red  slivers  or  lamellae  which  are  insoluble  in 
alcohol  and  ether,  but  dissolve  in  an  aqueous  or  alcoholic 
solution  of  caustic  potash  with  a  red-violet  color  which  soon 
goes  over  into  dark  yellow. 

8.    Pigment  of  Paxillus  atr ot oniento sus . 

197.  Thorner  (I)  has  extracted  a  pigment  from  the  above- 
named  fungus  which  is  deposited  in  crystalline  form  upon 


114  BOTANICAL   MICROTECHNIQUE. 

the  hyphc-K.  These  crystals  are  colored  brown  only  at  the 
surface  of  the  fungus,  those  in  its  interior  being  at  most 
pale  gray  or  yellowish.  In  the  air  the  colorless  crystals 
gradually  assume  a  brown  color.  According  to  Thorner, 
they  represent  a  hydroquinone-like  body  which  gradually 
passes  over  into  the  corresponding  quinone.  For  the  recog- 
nition of  the  quinone,  Bachmann  (II,  7)  recommends  strongly 
dilute  caustic  potash  or  soda^  which  instantly  dissolves  it  with 
a  greenish-brown  color. 

9.  Tannins. 

198.  All  those  substances  which  give  a  blue-black  or  green 
color  with  iron  salts  are  commonly  designated  as  tannic 
acids  or  tannins.  There  belong  here,  of  the  better  known 
compounds,  especially : 

Pyrocatechin,  C.HXOH),; 

Pyrogallic  acid,  C.H3(OH)3 ; 

Protocatechuic  acid,  C,H3.(OH),.COOH  ; 

Gallic  acid,  C,H,.(0H)3.C00H  ; 

Gallo-tannic  acid,  C,,H,„0,  (=  digallic  acid  ?)  ;  and  besides 
these  there  are  many  other  compounds  whose  constitution 
is  not  yet  certainly  determined  (cf.  Beilstein,  III,  431  ff.). 
We  have  no  trustworthy  methods  for  the  certain  micro- 
chemical  distinction  of  these  substances,  although  this  is 
the  more  to  be  desired  since  we  have  certainly  to  do  with 
very  different  .substances  and,  as  Reinitzer  (I)  has  lately 
shown,  it  is  very  hazardous  to  assume  a  common  physio- 
logical function  for  this  whole  group  of  compounds. 

But  a  detailed  account  of  the  methods  used  for  the  recog- 
nition of  the  whole  group  of  tannins  seems  to  be  demanded 
by  their  wide  distribution  in  plants.  The  following  reac- 
tions have  been  made  use  of  in  their  study : 

a.   Iron  Salts. 

199.  Of  the  iron  salts,  an  aqueous  solution  of  ferric  chlo- 
ride was  formerly  chiefly  used  ;  but  it  has  the  disadvantage 
that  it  has  nearly  always  an  acid  reaction,  and  when  used  in 


MICROCHEMIS  TR  V.  1 1 5 

excess  with  the  tannins  which  give  a  green  color  with  iron, 
it  very  quickly  stops  the  reaction.  H.  Moeller  (I,  LXIX) 
used,  however,  a  solution  of  anhydrous  ferric  cJilo^ide  in 
water-free  ether  as  a  reagent  for  tannin.  This  is  especially 
adapted  to  the  study  of  large  parts  of  plants,  such  as  whole 
leaves  or  pieces  of  them. 

Loew  and  Bokorny  (I,  370,  note)  have  recently  used  for 
the  recognition  of  tannin  in  algae  a  concentrated  aqueous 
solution  of  ferrous  sulphate^  in  which  the  algae  were  allowed 
to  be  exposed  to  the  air  for  from  twelve  to  twenty-four 
hours.  I  have  obtained  in  this  way  a  very  intense  reaction 
in  Spirogyra  and  Zygnema.  This  may  be  hastened  by  warm- 
ing to  60°  C. 

A  very  rapid  reaction  is  produced  hy  ferric  acetate,  accord- 
ing to  Moeller  (I,  LXIX),  in  the  form  of  the  concentration  of 
the  ofificinai  tincttira  ferri  acetici,  which  contains  about  ^% 
of  iron  or  about  20^  of  Fe3(C2H30aX. 

(^.  Cuprlc  Acetate. 

200.  Cupric  acetate,  which  was  introduced  into  micro, 
chemistry  by  Moll  (I),  has  the  advantage  that  it  forms  an 
insoluble  precipitate  with  tannins.  This  is  brownish  in 
color,  but  takes,  on  subsequent  treatment  with  iron  salts,  a 
blue  or  a  green  color  according  to  the  kind  of  tannin  con- 
cerned. Moll  places  the  tissues  to  be  studied  in  a  concen- 
trated aqueous  solution  of  cupric  acetate  and  leaves  them  in 
it  from  eight  to  ten  days,  or  as  much  longer  as  may  be  de- 
sired. Sections  prepared  from  this  material  are  treated  for 
a  few  minutes  with  a  5^  solution  of  ferric  acetate,  and,  after 
it  is  washed  out,  may  be  preserved  in  glycerine  or  glycerine- 
gelatine. 

If  it  is  desired  at  the  same  time  to  fix  the  cell-contents, 
one  may  use,  according  to  Klercker  (I,  8),  a  concentrated 
alcoholic  solution  of  cupric  acetate*  instead  of  the  aqueous 
solution.  The  pieces  of  tissue  should  be  left  several  days, 
at  least,  in  it. 

*  This  solution  must  be  kept  in  the  dark. 


1,6  BOTANICAL   MICROTECHNIQUE. 

y.   Potassium   Bichromate  and  Chromic  Acid. 

201.  Most  tannins  form  with  potassium  bichromate  a  volu- 
minous precipitate  which  is  bright  brown  or  blackish  brown 
according  to  the  quantity  of  tannin  present,  and  which  is 
insoluble  in  water  or  in  an  excess  of  the  salt,  and,  according 
to  Moeller  (I,  LXIX),  probably  consists  of  purpurogallin. 

Potassium  bichromate  is  most  conveniently  used  by  placing 
large  pieces  of  the  tissue  to  be  examined  for  one  or  several 
days  in  a  concentrated  aqueous  solution  of  this  salt,  and 
then  preparing  sections  after  washing  out  the  bichromate. 
These  sections  generally  show  the  precipitate  in  the  places 
where  the  tannin  was  formerly  present.  They  may  be  pre- 
served unchanged  in  glycerine  or  glycerine-gelatine.  Thicker 
sections  or  larger  fragments  may  also  be  transferred  to  Can- 
ada balsam.  But  of  course  they  must  first  be  dehydrated 
with  alcohol  and  cleared  with  clove-oil  or  the  like  (cf.  §§  14-22). 

The  precipitate  produced  by  potassium  bichromate  does 
not  change  its  color  with  iron  salts,  or,  as  Overton  (II,  5) 
has  shown,  with  sulphurous  acid.  Even  hydrogen  peroxide 
does  not  attack  it. 

To  obtain  a  rapid  reaction  J.  af  Klercker  (I,  8)  recom- 
mends for  many  cases  that  the  objects  be  plunged  in  a  boil- 
ing solution  of  potassium  bichromate.  In  fact  an  immediate 
reaction  may  thus  be  obtained  with  algae  and  sections  of 
higher  plants. 

According  to  Moeller  (I,  LXX),  the  diffusion  of  the  bichro- 
mate is  much  hastened  by  the  addition  of  a  few  drops  of 
acetic  acid. 

202.  Dilute  chromic  acid  of  about  i^  appears  to  give  a 
reaction  similar  to  that  of  potassium  bicarbonate,  and  may 
be  used,  as  well  as  mixtures  of  chromic  and  osmic  acids,  for 
the  recognition  of  tannins.  These  have  the  advantage  of 
fixing  the  cell-contents  well  at  the  same  time. 

But  it  should  be  noted  that,  according  to  Nickel  (I,  74),. 
various  compounds  not  related  to  the  tannins  give  similar 
precipitates  with  potassium  bichromate. 


MICR  O  CHE  Alls  TRY,  11/ 

6.   Osmic  Acid. 

203.  Osmic  acid  is  rapidly  reduced  by  tannic  acids  and 
very  soon  forms  with  them  a  solution  sometimes  bluish  and 
sometimes  brownish,  and  finally  a  black  precipitate.  For 
the  reaction  a  i^  solution  should  be  used.  The  osmic  acid 
is  regenerated  and  the  preparation  wholly  decolorized  by 
peroxide  of  hydrogen. 

If  hydrochloric  acid  be  first  added  to  the  preparation  and 
then  \io  osmic  acid,  there  appears,  according  to  Dufour 
(I,  3  of  separata),  in  a  few  minutes  a  blue  color,  and  soon,  if 
much  tannic  acid  be  present,  a  blue  precipitate. 

As  Overton  (II,  5)  has  shown,  albuminoids  saturated  with 
tannins  are  browned  ;  thus  he  obtained  a  beautiful  brown 
coloring  of  protein  crystalloids  on  leaving  sections  from  the 
endosperm  of  Ricinus,  from  which  the  fat  had  been  removed,, 
for  about  ten  minutes  in  a  dilute  solution  of  tannin,  and 
transferring  them,  after  careful  washing,  to  2</o  osmic  acid. 

€.   Ammonium  Molybdate. 

204.  Gardiner  (III)  used  for  the  recognition  of  tannins  a 
concentrated  solution  of  ammonium  molybdate  in  a  saturated 
ammonium  chloride  solution.  This  gives  with  most  tannins 
a  yellow  precipitate,  with  digallic  acid  a  red  one,  and  with 
gallic  acid  a  compound  soluble  in  ammonium  chloride  solu- 
tion. 

C.   Sodium  Tungstate. 

205.  Bramer  lately  recommends  (I)  for  the  recognition  of 
tannin  a  solution  of  i  gram  of  sodium  tungstate  and  2  grams 
of  sodium  acetate  in  10  ccm.  of  water.  This  should  give  a 
brown  precipitate  with  gallic  acid,  and  a  reddish-yellow  one 
with  gallotannic  acid.  But  the  presence  of  tartaric  or  citric 
acid  hinders  the  reaction. 

rj.   Alkaline  Carbonates. 

206.  Alkaline  carbonates  cause  the  precipitation,  in  cells 
containing  tannin,  of  globular  or  rod-shaped  bodies  which,, 
when  freshly  precipitated,  may  be  redissolved  on  washings 


tig  BOTANICAL   MICROTECHNIQUE. 

out  the  carbonate.  But  after  a  time  they  lose  this  capacity 
and  are  gradually  transformed  into  solid  and  brittle  bodies. 
The  same  precipitates  are  formed  when  tannin  and  ammo- 
nium carbonate  are  brought  together  in  a  test-tube ;  but  if 
the  carbonate  solution  be  very  dilute,  it  only  occurs  when  a 
gradual  accumulation  of  this  salt  is  made  possible  ;  which 
Klercker  (I,  42)  accomplished  by  placing  the  tannin  solution 
in  a  capillary  tube  sealed  at  one  end,  and  then  putting  the 
whole  in  a  large  vessel  filled  with  the  carbonate  solution. 
Since,  according  to  Klercker's  researches,  these  precipitates 
have  been  seen  only  in  cells  containing  tannin  and  always 
give  the  tannin  reactions  with  potassium  bichromate  and 
other  reagents,  it  would  appear  that  the  alkaline  carbonates 
may  well  be  used  as  reagents  for  tannin. 

But  not  all  tannins  are  precipitated  by  them  ;  for  instance, 
gallic  acid  is  not. 

207.  In  order  to  carry  out  the  reaction  described,  sections 
of  the  plants  to  be  studied  are  placed  on  the  slide  in  a  drop 
of  a  1-5^  solution  of  an  alkaline  carbonate  or  the  plants 
are  cultivated  for  some  time  in  a  very  dilute,  about  .02J^, 
solution.  The  carbonates  of  potassium,  sodium,  and  ammo- 
nium seem  to  be  equally  active,  but  the  ammonium  salt  has 
been  most  used.  Ammonium  chloride  seems  also  to  act  in 
the  same  way;  but  the  bicarbonates  produce  no  precipitate. 

A  very  suitable  object  for  study  is  found  in  the  stem  or 

petiole    of    Ricimis    communis, 


]^^^  ^  ^^  «3.  #««« F  which  contain  red  pigment-cells 
•%  J^SLJ^  ^\%  1^  4  in  the  pith  and  bark.  If  longi- 
^*.#^%^JTl  ^  1m       tudinal   sections    of    these    are 


•]|  —     placed  in  a  i^  ammonium  car- 

Fic.  30. -Cells  from  the  pith  of  the  p€ti-  bonatc   solution,   granular   pre- 

o\eoi  RictMUs communis.    The  section        '     '.     .  •  i         i.  ^• 

had  Iain  an  hour  in  a  i%  solution  of    ClpltatCS  appear  m  a  short  tUTlC, 
ammonium  carbonate.  l'i  j       n  n       ^  ^  ^i 

which  gradually  collect  together 
and,  after  about  an  hour,  have  drawn  almost  the  entire  pig- 
ment to  themselves  (cf.  Fig.  30).  Spirogyra  cells  which 
contain  tannin  also  form  excellent  objects  for  study,  since  a 
precipitate  is  almost  immediately  formed  in  them  by  ammo- 
nium carbonate. 


MICR O CHEMIS  TR  V.  1 1 9 

'&,    Live-staining  with   Methylene   Blue. 

208.  As  was  shown  by  Pfeffer  (II,  186),  methylene  blue  is 
accumulated  by  tannin-bearing  cells.  In  a  solution  of  this, 
pigment  the  cell-sap  which  contains  tannin  first  takes  an 
evidently  blue  color,  and  then  there  usually  occur  within 
these  cells  deep-blue  precipitates  of  different  forms,  which 
consist  of  a  compound  of  tannin  and  methylene  blue  and 
may  finally  remove  all  pigment  from  the  cell-sap. 

This  reaction  seems  to  take  place  in  all  tannin-bearing- 
cells,  and  is  especially  valuable  because  it  can  be  conducted 
on  the  living  cells,  and  without  the  diminution  of  their 
vitality.  For  its  application  a  solution  should  be  used 
which  contains  one  part  of  methylene  blue  in  500,000  parts- 
of  filtered  rain-water.  In  this  the  tissues  are  left  for  from 
one  to  twenty-four  hours.  Of  course  a  large  quantity  of 
fluid  must  be  used  to  allow  an  abundant  accumulation  of 
coloring  matter. 

But  methylene  blue  is  accumulated  by  other  substances 
than  tannins  ;  according  to  Waage  (I,  253),  by  phloroglucin. 

10.  Alkaloids. 

209.  Under  the  name  alkaloids  is  included  a  large  group 
of  natural  basic  compounds  which  contain  nitrogen  and 
show  a  certain  agreement  in  many  chemical  reactions. 

They  are  all  precipitated  hy phospJio-molybdic  acid,  which 
may  conveniently  be  used  in  a  10^  aqueous  solution.  But 
this  reaction  cannot  be  generally  used  in  microchemistry, 
since  this  acid  precipitates  many  other  compounds,  e.g.  the 
proteids.  But,  according  to  Errera  (V),  these  may  be  dis- 
tinguished from  the  alkaloids  by  extracting  the  alkaloids 
from  a  part  of  the  sections  to  be  studied  before  treatment 
with  phospho-molybdic  acid,  while  the  proteids  are  com- 
pletely precipitated.  Errera  found  especially  adapted  for 
this  use  alcohol  which  contains  in  each  20  ccm.  a  gram  of 
crystallized  tartaric  acid.  He  allows  this  to  act  upon  the 
sections  from  half  an  hour  to  twenty-four  hours,  when  a 
complete    solution    of   the    alkaloids    is    effected,   with    the 


1 20  BO  TA NICA L   MICRO  TECIINIQ UE. 

simultaneous  precipitation  of  the  proteids.  If  no  precipi- 
tation  takes  place  in  the  sections  so  treated  on  the  addition 
of  phospho-molybdic  acid,  while  it  does  occur  in  the  untreated 
sections,  one  may  deduce  the  presence  of  alkaloids-  in  the 
objects  concerned,  provided  that  these  were  the  only  sub- 
stances soluble  in  tartaric-acid-alcohol  which  are  precipitated 
by  phospho-molybdic  acid.  But  this  is  by  no  means  the 
case  (cf.  Molisch,  I,  15),  and  the  above  reactions  can  give 
reliable  results  only  in  very  special  cases. 

The  same  is  true  for  the  other  group-reactions  for  the 
alkaloids;  but  the  value  of  the  special  reactions  for  the 
different  alkaloids,  to  which  we  now  pass,  is  so  much  the 
greater. 

a.  Aconitine,  CgaH^NO,^. 

210.  According  to  Errera,  Maistriau  and  Clautriau  (I)  a 
solution  of  iodine  and  potassium  iodide  is  best  adapted  for 
the  microchemical  recognition  of  aconitine,  with  which  it 
forms  a  red-brown  precipitate.  These  authors  also  obtained 
a  carmine-red  color  in  presence  of  aconitine  with  sidphnric 
/icid  diluted  with  \  ox  \  oi  its  volume  of  water,  especially 
after  the  moistening  of  the  preparations  with  a  solution  of 
cane-sugar. 

b.  Atropine,  C^H^NO,. 

211.  De  Wevre  (I)  used  a  solution  of  iodine  and  iodide  of 
potassium  for  the  microchemical  recognition  of  atropine,  in 
cells  containing  which  a  brown  precipitate  is  produced. 
After  some  time  star-shaped  crystallizations  with  a  metallic 
lustre  appeared.  Phospho-molybdic  acid,  which  gives  a 
yellow  precipitate,  is  also  applicable  in  many  cases. 

c,  Berberin,  C„H.,NO,  +  4jH,0. 

212.  Berberih  occurs,  according  to  the  consonant  state- 
ments of  Naegeli  and  Schwendener  (I,  503),  O.  Herrmann 
(I,  14),  and  Rosoll  (I,  20),  in  the  young  cells  of  Berberis 
vulgaris  as  a  golden-yellow  fluid  in  the  cell-cavity ;  while  in 


MICROCHEMIS  7 'A'  Y.  121 

the  older   parts  it  incrusts   especially  the  cell-walls  of  the 
xylem. 

For  its  microchemical  recognition  O.  Herrmann  treats 
the  sections  first  with  alcohol  and  then  adds  dilute  nitric 
ncid  (one  part  of  acid  to  about  fifty  parts  of  water).  The 
golden-yellow  color  passes  over  at  once  into  brownish  yellow, 
and  soon  golden  -  yellow 
star-shaped  crystals  are  de- 
posited, while  the  cell-sap 
becomes  gradually  color- 
less. The  same  crystals  of 
berberin  nitrate  maybe  ob-  pio.  si.-Groups  of  crystals  of  berbenn  ni- 
tained  by  placing  the  sec-  S^s  ottrb\'rk  Jf ^irJ.?,^^^^^^ 
tions  directly  in  dilute  nitric  '°^""°"  °'  "'^"^  "^'^• 
acid.  I  use  2  parts  of  the  officinal  nitric  acid  in  loo  parts 
of  water.  The  berberin  nitrate  separates  out  in  the  form 
of  clustered  crystals  (cf.  Fig.  31)  in  the  interior  of  the  ber- 
berin-bearing  cells.  These  crystals  are  strongly  doubly 
refractive  and  so  far  pleochroic  (§  356,  note)  that  they 
appear  quite  colorless  in  a  certain  position  of  the  nicols, 
while  after  rotation  through  90°  they  appear  deep  golden 
yellow. 

Herrmann  also  used  ammonium  sulphide,  which  gives  a 
brownish  color  with  it,  for  the  recognition  of  berberin. 

Rosoll  used  a  solution  of  iodine  and  potassium  iodide  for 
the  same  purpose.  This  is  added  to  the  sections  in  small 
quantity  after  preliminary  treatment  with  alcohol.  There 
are  then  formed  very  characteristic  hair-like  crystals,  ar- 
ranged in  tufts,  which  are  green  or,  in  the  presence  of  large 
quantities  of  the  reagent,  yellowish  or  reddish  brown,  and 
are  soluble  in  sodium  hyposulphite.  The  crystals  thus 
obtained  cannot  be  preserved  in  Canada  balsam,  according 
to  my  experiments. 

If  hydrochloric  acid  be  added  to  the  yellow  aqueous  ex- 
tract of  the  dried  bark,  there  are  formed,  according  to 
Naegeli  and  Schwendener  (I,  504),  numerous  yellow  and 
often  radially  grouped  needle-like  crystals  of  berberin  chlo- 
ride. 


122  BOTANICAL   MICROTECHNIQUK. 

d.  Brucine,  C„H„N,0,  +  4H,0. 

213.  For  the  recognition  of  brucine,  which  accompanies 
strychnine  in  the  seeds  of  Strychtios  nux-vomica,  Lindt  (11^ 
239)  used  a  mixture  of  five  drops  of  selenic  acid,  of  specific 
gravity  1.4,  and  one  or  two  drops  of  nitric  acid,  of  specific 
gravity  1.2.  He  allowed  this  to  run  under  the  cover-glass 
to  thin  sections  whose  fat  had  been  extracted  by  petroleum- 
ether.  The  stratified  cell-walls  then  quickly  became  bright- 
red,  but  gradually  changed  to  orange  and  yellow,  while  the 
cell-contents  remained  colorless.  Lindt  concluded  from  this 
that  only  the  walls  contain  brucine  (cf.  also  §  219). 

e.  Colchicine,  C„H„NO,. 

214.  Colchicine  occurs,  according  to  O.  Herrmann  (I,  18)^ 
in  the  corm  of  Colchicum  mitiimnale,  in  the  contents  of  two 
or  three  rows  of  cells  which  immediately  surround  the 
vascular  bundle  and  are  distinguished  from  the  neighboring 
parenchyma-cells  by  being  free  of  starch.  For  the  recogni- 
tion of  colchicine  this  author  used  ammonia,  which  turns  it 
a  deep  yellow. 

Errera,  Maistriau  and  Clautriau  (I)  have  lately  character- 
ized it  as  giving  a  yellow  color  with  siilpJiiiric  acid  diluted 
with  two  or  three  volumes  of  water,  a  brown-violet  with  sul- 
phuric and  nitric  acids,  a  brown  with  iodine,  and  a  yellowish 
precipitate  with  potassic-merctiric  iodide  and  hydrochloric 
acid. 

f.   Corydalift,  C,«H,,NO,. 

215.  According  to  Zopf  (VI,  113),  corydalin  shows  the 
following  reactions  :  It  is  precipitated  from  aqueous  solu- 
tions of  its  salts  by  caustic  alkalies  and  alkaline  carbonates, 
but  is  redissolved  in  an  excess  of  the  former.  A  brown 
precipitate  is  produced  in  solutions  of  its  salts  by  solutions 
of  iodine  or  of  iodine  and  potassium  iodide,  a  yellow  one  by 
potassium  chromate,  a  white  one  by  mercuric  chloride,  a 
yellow  one  by  gold  and  platinum  chlorides  and  by  sodium 
metatungstate,    a   yellowish-white    one    by  potassic-mcrcuric 


MICROCHEMIS  TRY.  1 2  3 

cJiloridc,    a   white    one    by  potassium   stilpJLo-cyanide.     The 
solution  in  spirit  is  precipitated  by  strong  alcohol. 

For  its  microchemical  recognition  Zopf  used  especially 
ammonia,  which  produces  a  dark  gray,  granular  precipitate 
in  the  cells  containing  corydalin,  and  iodine  with  iodide  of 
potassium,  which  causes  a  deep  red-brown  precipitate.  Picric 
acid  also  causes,  according  to  Zopf,  a  readily  recognizable 
precipitate  in  corydalin-bearing  cells. 

g.  Cytisine,  C,oH,,N30. 

216.  Cytisine  occurs,  according  to  Rosoll  (I,  24),  in  all  the 
organs  of  Cytisiis  Labnrnnm,  but  most  abundantly  in  the 
contents  of  the  cells  of  the  ripe  seed.  This  author  uses  the 
following  reactions  for  its  recognition  : 

Iodine  and  iodide  of  potassium  give,  even  when  very  dilute^ 
a  brownish  color  and  then  a  dark  red-brown  precipitate, 
soluble  in  sodium  hyposulphite. 

Picric  acid,  added  to  thin  sections,  produces  in  a  short 
time  scale-like  crystal-groups  of  a  reddish-yellow  color. 

Concentrated  sulpJiuric  acid  dissolves  cytisine  with  a  bright 
reddish-yellow  color ;  if  very  small  bits  of  solid  potassium 
bichromate  be  added  to  this  solution,  it  becomes  first  yellow,, 
then  brown,  and  finally  green. 

Phospho-molybdic  acid  produces  immediately  a  yellow  tur- 
bidity in  the  sections. 

h.  Opium  Alkaloids. 

217.  According  to  the  investigations  of  Clautriau  (I), 
several  of  the  so-called  opium  alkaloids  occur  in  the  latex 
of  the  living  plant  in  Papaver  somniferum. 

I.  Morphine,  C17H19NO3. 

The  presence  of  morphine  is  recognized  by  this  author 
by  the  fact  that  the  latex  gives  precipitates  with  iodine  and 
potassium  iodide,  potassium-bismuth  iodide,  potassium-calcium 
iodide,  potassic-mercuric   iodide,  and  phospho-molybdic   acid  - 


124  BOTANICAL   MICROTECHNIQUE. 

that  it  reduces  iodic  acid;  and  that  it  is  colored  red-brown 
by  a  2%  solution  of  titanic  acid  in  sulphuric  acid,  or  deep 
violet  by  a  solution  containing  five  drops  of  methylal 
(CH,(OCH,),)  to  a  ccm.  of  concentrated  sulphuric  acid. 

2.   Narcotine,  CasHasNOT. 

Clautriau  deduces  the  presence  of  narcotine  in  the  latex 
from  its  becoming  colored  reddish  orange  with  a  solution  of 
sodium  selenate  in  sulphuric  acid,  as  also  happens  with  mixt- 
ures of  morphine  and  narcotine.  Besides,  it  gives  a  precipi- 
tate with  palladotis  chloride  and  with  iridoiis  chloride,  while 
morphine  and  codeine  give  no  precipitate  with  the  former, 
and  but  a  slight  one  with  the  latter  reagent. 

3.  Narcelne,  CasHaeNOg. 

According  to  Clautriau,  a  yellow  color  following  the  addi- 
tion of  the  solution  of  methylal,  mentioned  under  ''  Mor- 
phine," indicates  the  presence  of  narceine  in  the  latex. 

i,  Piperine,  C^H^NO,. 

217a.  Piperine,  which  has  been  recognized  in  the  fruits  of 
various  Piperacece,  is  slightly  soluble  in  boiling  water,  more 
easily  soluble  in  alcohol  than  in  ether,  readily  soluble  in 
benzol,  insoluble  in  dilute  acids. 

For  its  microchemical  recognition  concentrated  sulphuric 
acid  may  be  used.  This  dissolves  piperine  with  a  yellow 
color  which  later  becomes  dark  brown,  and,  after  20  hours, 
greenish  brown  (Husemann  I,  491).  But,  since  various  other 
substances  give  the  same  reaction,  it  can  only  be  of  value  as 
negative  evidence  through  its  failure  to  appear. 
:  We  owe  to  Molisch  (I,  27)  a  very  useful  method  of  recog- 
nizing piperine  microchemically.  A  drop  of  alcohol  is  first 
placed  on  the  sections  on  the  slide  and  dissolves  the  piperine. 
Then  the  sections  are  covered  with  a  cover-glass,  and  the 
alcohol  is  allowed  to  evaporate  until  it  occupies  only  about  a 
quarter  of  the  space  under  the  cover.  Then  water  is  added 
and  causes  at  once  a  strongly  milky  turbidity,  if  sections  of 


MICR  0  CHEMIS  TR  Y.  1 2  5 

the  pepper-fruit  are  used.  This  turbidity  is  due  chiefly  to 
the  yellow  resin  which  is  also  dissolved  in  the  alcohol. 
After  a  time  (about  a  quarter  of  an  hour)  there  are  seen 
characteristic  colorless  crystals,  especially  abundant  at  the 
edge  of  the  cover-glass,  which  have  very  commonly  an  ap- 
proximately sabre-shaped  outline,  but  are  not  seldom  grown 
together  in  most  various  ways, 
as  is  shown  in  Fig.  32,  which  rep- 
resents crystals  obtained  by  the 
method  described.  All  these 
crystals  give  the  piperine  reac- 
tions above  described  and  un- 
doubtedly consist  of  that  sub- 
stance. 

Similar   piperine    crystals    are 

formed,     according      to       Molisch  F.g.  s^-Crystals  of  piperine. 

{I,  28)»  within  or  in  the  vicinity  of  the  piperine-cells  of  pep- 
per, if  thin  sections  are  placed  under  a  cover-glass  in  water 
or  glycerine  and  kept  in  a  moist  chamber  for  several  hours. 
""  Sections  which  are  pressed  and  rubbed  under  a  cover-glass 
in  water  show  piperine  crystals  within  the  first  quarter  of  an 
hour." 

k.  Sinapine,  CieH^NO^. 

218.  This  occurs  in  the  seeds  of  white  mustard  as  sinapine 
sulphocyanide  (C,eH3,NO,.HCNS). 

According  to  Molisch  (I,  31),  it  is  best  recognized  with  a 
concentrated  solution  of  caustic  potash.  Sections  of  white 
mustard  seeds  placed  in  this  become  at  once  yellow,  and,  on 
warming,  deep  orange.  But  this  reaction  has  a  very  limited 
value,  since  the  glucoside  sinalbine,  also  found  in  white 
mustard-seeds,  becomes  yellow  with  caustic  potash. 

/.   Strychnine,  QiH^^N^O^. 

219.  Strychnine  is  dissolved  without  color  by  a  concen- 
trated sulphuric  acid ;  but  if  solid  potassium  bichromate  be 
added  to  the   solution,  a  beautiful   violet   color  appears  at 


12(3  BOTANICAL   MICROTECHNIQUE. 

once.  This  reaction  has  been  used  by  Rosoll  (I,  17)  for  the 
microchemical  recognition  of  strychnine.  He  phiced  thin 
sections  from  the  seeds  of  StrycJinos  nux-vofnica  first  in  con- 
centrated sulphuric  acid,  and  observed  that  the  contents  of 
the  endosperm-cells  become  plainly  rose-red  from  the  pres- 
ence of  proteids  and  sugar  (cf.  §  227),  except  the  oil-drops, 
which  remained  uncolored.  If  now  a  small  fragment  of 
potassium  bichromate  be  added,  the  previously  colorless  oil- 
drops  take  a  beautiful  violet  color.  Rosoll  concludes  from 
this  that  the  strychnine  is  dissolved  in  the  oil-drops. 

Lindt  (II)  used  a  solution  of  an  excess  of  ccric  sulphate  in 
concentrated  sulphuric  acid  for  the  recognition  of  strych- 
nine. In  spite  of  the  gradual  reddening  of  the  eerie  oxide, 
this  reagent  remains  fit  for  use  a  long  time.  Before  its 
use  the  sections  should  be  treated  with  petroleum-ether 
and  alcohol  for  the  removal  of  fatty  oils,  grape-sugar,  and 
brucine.  If  the  solution  be  then  added  to  the  sections,  all 
the  cell-walls  become  at  once  colored  more  or  less  strongly 
violet-blue,  while  the  contents  of  the  cells  at  first  remain 
colorless.  But  after  some  time  the  reaction  is  disturbed  by 
various  circumstances.  Lindt  concludes  from  this  observa- 
tion that  the  strychnine  is  contained  in  the  cell-walls.  But 
Rosoll  (I,  18)  expresses  the  opinion  that,  during  the  extrac- 
tion with  petroleum-ether,  the  strychnine,  formerly  dissolved 
in  the  fatty  oil,  is  removed  with  it  and  becomes  partly 
diffused  into  the  cell-walls. 


;//.    Theobromine,  Dime tliyl-x ant Jiin,  C^HgN^Oj. 

220.  The  alkaloid  contained  in  the  cocoa-bean,  theobro- 
mine, is  best  recognized,  according  to  Molisch  (I,  23),  by 
means  of  gold  chloride^  by  adding  to  sections  upon  a  slide, 
first  a  drop  of  concentrated  hydrochloric  acid  and  then, 
after  about  a  minute,  a  drop  of  a  3^  solution  of  gold  chlo- 
ride. As  soon  as  a  part  of  the  fluid  has  evaporated,  long 
yellow  needles  are  formed  at  the  Q(\gQ  of  the  drop,  and 
finally  unite  into  feathery  or  tufted  groups.     The  crystals 


MICROCHEMIS  TRY.  12/ 

-consist   of  the   double  chloride   of  gold   and  theobromine, 
C,H,NA.HCl.AuCl3. 

n.   Caffeine,  Theine,  MetJiyl-theobromine,  Trimethyl-xanthiriy 
QH.^N^O,  +  H,0. 

221.  Molisch  recommends  (I,  'j^  gold cJiloride  for  the  micro- 
chemical  recognition  of  caffeine.  It  is  used  in  the  same 
way  as  for  theobromine,  and  there  are  formed  tufted  ra- 
diating needles  of  the  composition  CgHjoN^O^.HCl.AuCl, , 
which  cannot  certainly  be  distinguished  from  those  of  the 
corresponding  theobromine  compound. 

Hanausek  (I)  has  lately  called  attention  to  the  fact  that, 
especially  when  the  gold  chloride  solution  contains  more 
than  3^  of  the  salt,  a  drop  of  it  with  concentrated  hydro- 
chloric acid  will,  in  any  event,  form  yellow  crystals  on  evap- 
oration. But  these  are  distinguished  from  the  caffeine- 
gold  chloride  crystals  by  the  fact  that  they  never  form 
sharp-pointed  or  tufted  needles,  like  those  of  the  caffeine- 
gold  compound,  but  consist  partly  of  vfery  short  crystals 
arranged  in  zigzag,  and  partly  of  very  long,  delicate,  yellow, 
rod-like  prisms  and  of  plates  with  rectangular  projections. 

Molisch  gives  also  another  method  for  recognizing  caf- 
feine. According  to  this,  sections  are  warmed  on  the  slide 
in  a  drop  of  distilled  water  until  it  bubbles,  then  allowed  to 
dry  at  the  ordinary  temperature,  and  the  residue  is  taken 
up  with  a  drop  of  benzol.  On  the  evaporation  of  the  benzol, 
the  caffeine  is  deposited  at  the  edge  of  the  drop  in  the  form 
of  numerous  colorless  needles. 

0,    Veratrine,  Cg.H^gNOn. 

222.  Veratrine  occurs,  according  to  Borscow  (I,  58),  in 
the  subterranean  parts  of  Veratrum  album,  especially  in  the 
walls  of  the  cells  of  the  epidermis  and  of  the  bundle-sheath 
(endodermis).  This  author  used  for  the  recognition  of  vera- 
trine a  mixture  of  one  part  of  concentrated  sulphuric  acid 
with  two  parts  of^water,  in  which  the  sections  to  be  exam- 


128  BOTANICAL   MICROTECHNIQUE. 

ined  are  directly  placed.  The  parts  containing  veratrine 
then  become  first  yellow,  then  reddish  orange,  and  finally 
red-violet. 

p.  Xanthine,  CsH.N.O,. 

222a.  Xanthine  has  been  recognized  by  Belzung  (II,  49) 
in  considerable  quantity  in  the  seedlings  of  Cicer  arietinum. 
It  crystallizes  out  in  the  interior  of  the  cells  of  plants  placed 
in  glycerine. 

II.  Nitrogenous  Bases. 

Nicotine,  C,oH,,N,. 

223.  The  following  reactions  have  been  recommended  for 
the  recognition  of  nicotine  by  Errera,  Maistriau  and  Clau- 
triau  (I) :  Phospho-tungstic  acid  gives  a  heavy  precipitate,  at 
first  yellow,  then  yellowish  green  ;  mercuric  chloride,  a  white 
one,  soluble  in  an  excess  of  ammonium  chloride  with  heat ; 
potassic-mercuric  iodide,  an  abundant  white  one  ;  platinnni 
chloride,  a  yellowish-white  one,  soluble  at  70°  C. ;  iodine  and 
potassium  iodide,  a  brownish-yellow  one  which  disappears 
later. 

12.  The  Proteids  and  Related  Compounds. 

224.  Although  the  albuminoid  substances  or  proteids 
undoubtedly  belong  to  the  most  important  constituents  of 
the  plasma-body  of  the  cell,  we  still  know  very  little  of  their 
chemical  constitution.  But,  at  all  events,  we  have  here  to 
do  with  a  group  of  dissimilar  compounds,  and  already  various 
investigators  have  distinguished  a  large  number  of  different 
proteids.  But  since  no  classification  of  the  proteids  has  yet 
found  general  acceptance,  and  especially  since  the  exact 
microchemical  distinction  of  the  proteids  which  are  recog- 
nized macrochemically  is  not  yet  possible,  it  seems  best  not 
to  discuss  these  researches  here ;  and  so  much  the  more  so 
since  they  have  not  led  to  any  morphologically  or  physio- 
logically interesting  results,  so  far  as  concerns  the  vegetable 
organism.     But  it  seems  important  to  briefly  collate  the 


MICROCHEMISTR  V.  1 29 

methods  heretofore  used  for  the  microscopical  recognition 
of  the  proteids  in  general,  and  then  to  describe  a  few  related 
substances  which  have  been  microscopically  recognized  in 
the  protoplasm. 

a.  Reactions  of  the  Proteids, 

a.  Solutions  of  Iodine. 

224a.  With  iodine  the  proteids  take  a  yellow  or  brown 
color,  according  to  the  strength  of  the  solution,  and  this 
reaction  has  been  much  used  for  their  recognition,  although 
many  other  substances  give  the  same  reaction.  But  ia 
many  cases  the  behavior  of  a  doubtful  body  with  iodine 
may  give  a  clew  to  its  nature.  It  is  best  to  use  a  solution 
of  iodine  and  potassium  iodide  containing  more  iodine  than 
is  used  for  the  recognition  of  starch,  since  iodine  is  taken  up 
much  less  freely  by  proteids  than  by  starch. 

ft.  Nitric  Acid. 

225.  Ordinary  concentrated  nitric  acid  gives  a  yellow 
color  with  proteids  by  the  formation  of  the  so-called  xayitho- 
proteic  acid.  This  reaction  may  be  hastened  by  gentle  warm- 
ing. The  color  becomes  considerably  deeper  by  the  addition 
of  caustic  potash  or  ammonia,  since  the  xanthoproteic  salts 
of  potassium  and  ammonium  are  more  deeply  colored  than 
the  free  acid.  But  this  reaction  is  not  entirely  trustworthy,, 
since  not  only  do  tyrosin  and  various  oxy-aromatic  com- 
pounds give  the  same  reaction,  but  also  certain  oils,  resins, 
and  alkaloids  (cf.  Nickel  I,  17). 

y.   Mill,on's  Reagent. 

226.  The  so-called  Millon's  reagent  is  a  mixture  of  iner- 
curic  and  mercuroiis  nitrates  and  nitrous  acid.  It  is  best  pre- 
pared, according  to  Plugge  (I),  by  dissolving  one  part  by 
weight  of  mercury  in  two  parts  of  nitric  acid  of  specific 
gravity  1.42,  and  then  diluting  it  with  twice  its  volume  of 
water.  According  to  Nickel  (I,  7),  it  may  be  prepared  by 
dissolving  i  ccm.  of  mercury  in  9  ccm.  of  concentrated  nitric 


130  BOTANICAL    MICROTECHNIQUE. 

acid  of  Specific  gravity  1.52  and  diluting  the  solution  with 
an  equal  volume  of  water.  The  reagent  so  obtained  becomes 
decomposed  in  time,  and  may  then  be  restored,  according  to 
Krasser  (I,  140),  by  the  addition  of  a  few  drops  of  a  solution 
of  potassium  nitrite. 

Millon's  reagent  gives  with  proteids  a  brick-red,  or  more 
rose-red,  color,  which  appears  usually  after  some  time  in  the 
cold,  but  much  more  quickly  on  gentle  warming,  without 
any  solution  of  the  proteids.  The  protein  crystalloids  in 
the  endosperm  of  RicimiSy  for  example,  retain  their  form 
unchanged  even  on  heating  nearly  to  the  boiling  point. 
This  reaction  is  also  very  delicate,  but  it  has  the  disadvan- 
tage that  it  takes  place  also  with  a  large  number  of  other 
compounds,  in  the  same  way;  according  to  Plugge  (I),  with 
all  those  that  contain  an  OH-group  on  the  benzol  nucleus. 

227.  Reactions  similar  to  that  of  Millon's  reagent  are 
given  by  Hofinamis  reagejit^  which  is  a  solution  of  mercuric 
nitrate  with  traces  of  nitrous  acid,  and  by  the  so-called 
Plugge  s  reagent,  which  consists  of  mercurous  nitrate  with 
traces  of  nitrous  acid  (cf.  Nickel  I,  12  and  13). 

5.   Raspail's  Reagent. 

227a.  The  so-called  Raspail's  reagent  consists  of  a  con- 
centrated aqueous  solution  of  cane-sugar  and  concentrated 
sulphuric  acid,  which  are  added  to  the  objects  to  be  tested 
at  the  same  time.  Proteids  are  colored  by  this  rosy-red  or 
somewhat  violet.  But  the  reaction  does  not  succeed  with 
all  proteids  and  does  occur  with  various  other  substances. 
Many  glucosides  and  alkaloids  are  colored  red  by  sulphuric 
acid  alone  (cf.  Nickel  I,  37  ff.). 

€.  Copper  Sulphate  and  Caustic  Potash. 

228.  Most  proteids  give  a  violet  reaction  on  treatment 
with  copper  sulphate  and  caustic  potash  ;  but  the  reaction 
is  neither  always  very  sharp  nor,  when  it  succeeds,  positive 
proof  of  the  presence  of  proteids.  The  reaction  may  be 
conducted  in  the  same  way  as  for  cane-sugar  (cf.  §  121). 


MICKOCHEMIS  TKY.  1 3 1 

This  reaction  was  used  on  Spirogyra  by  Loew  and  Bokorny 
(I,  194)  by  first  placing  the  plant  in  a  ,2%  caustic  potash 
solution  for  half  an  hour  to  an  hour,  then,  after  washing-,  in 
a  10^^  solution  of  copper  sulphate  for  an  hour,  and  finally, 
after  repeated  washing  on  the  slide,  moistening  with  a  2  ^ 
■caustic  potash  solution. 

^.     Alloxan.    M  e  s  o  x  a  1  y  1  u  r  e  a  ,    C4HaN804. 

229.  Alloxan  has  lately  been  proposed  by  Krasser  (I,  18) 
as  a  reagent  for  proteids.  It  is  added,  preferably  to  pre- 
viously dried  sections,  in  concentrated  aqueous  or  alcoholic 
solution.  It  then  colors  most  proteids  purple-red,  and,  ac- 
cording to  Krasser,  this  color  is  not  changed  by  concentrated 
caustic  soda.  But  the  reaction  is  hindered  by  free  acids. 
Of  the  other  organic  substances  which  Krasser  has  treated 
in  tlie  same  way,  unfortunately  without  enumerating  them 
in  his  publication,  only  tyrosin,  asparagin,  and  aspartic 
acid  gave  the  same  reaction  ;  and  it  therefore  seems  to  be 
caused  by  the  molecular  group  common  to  these  substances, 
CH,.CHNH,.COOH. 

But,  as  Klebs  has  shown  (^I V,  699),  the  valine  of  this  reac- 
tion is  very  slight ;  for  various  inorganic  compounds  like 
phosphates  and  bicarbonates  of  the  alkalies  give  a  very  deep 
red  color  with  alloxan,  and  alloxan  itself  turns  red  on  evap- 
oration in  the  air ;  and  this  color,  as  well  as  that  produced 
with  proteids,  is  changed  to  violet  by  caustic  soda,  according 
to  Klebs. 

rf.     Aldehydes. 

229a.  Various  aldehydes,  especially  salicylic  aldehyde^ 
€,H,OH.CHO,  anisic  aldehyde,  C,H,.OCH,.CHO,  vanillin, 
C„H3.0H.OCH3.CHO,  and  cinnamic  aldehyde.  C,H,.CH  : 
CH.CHO,  have  lately  been  recommended  for  the  micro- 
chemical  recognition  of  albuminoids  by  Reichl  and  Mikosch 
(I,  34).  The  objects  to  be  tested  are  first  left  for  24  hours 
in  a  \-\^  alcoholic  solution  of  the  aldehyde  used,  and  then 
placed  on  the  slide  in  a  mixture  of  equal  volumes  of  water 
and  sulphuric  acid,  to  which  a  few  drops  of    ferric  sulphate 


132  BOTANICAL   MICROTECHNIQUE. 

(Fe,(SO,),)  have  been  added.  Most  proteids  then  show, 
either  at  once  or  after  some  time,  a  coloration  depending  not 
only  on  the  nature  of  the  aldehyde  used,  but  also  varying; 
with  the  different  albuminoids,  and  not  appearing  at  all,  with 
many.  The  colors  with  the  salicylic  and  anisic  aldehydes 
and  vanillin  vary  between  red,  violet,  and  blue  ;  while  the 
cinnamic  aldehyde  produces  yellow  or  orange-yellow  colors, 
with  proteids. 

Salicylic  aldehyde  has,  according  to  the  statements  of  the 
authors  mentioned,  the  advantage  of  more  completely  fixing 
the  proteids  and  of  making  them  more  resistent  to  the  dis- 
solving power  of  sulphuric  acid. 

0.    Yellow    prussiate   and   ferric    chloride. 

230.  Zacharias  (I,  211)  has  lately  reintroduced  into  micro- 
chemistry  a  method  of  recognizing  proteids  first  used  by  Th. 
Hartig,  which  may  be  here  referred  to,  although  it  is  more 
a  staining  process  than  a  chemical  reaction.  This  test  was 
carried  out  by  Zacharias  by  placing  the  tissues  to  be  tested 
for  an  hour  in  a  mixture  of  one  part  of  a  10^  aqueous  solu- 
tion of  potassiuin  ferrocyanide^  one  part  of  water  and  one 
part  of  acetic  acid  of  specific  gravity  1.063.  This  mixture,, 
which  must  always  be  freshly  prepared,  on  account  of  its 
ready  decomposition,  is  now  washed  out  with  60^  alcohol 
until  the  washing  fluid  no  longer  gives  an  acid  reaction  or  a 
blue  color  with  ferric  chloride.  Then  a  dilute  solution  of 
ferric  chloride  is  added,  which  causes  a  deep  blue  coloring  of 
the  albuminoids  (Berlin  blue)  inconsequence  of  the  ferrocy- 
anide  retained  by  them. 

231.  To  recognize  albumen  in  the  cytoplasm  of  Spirogyra^ 
Loew  and  Bokorny  (I)  first  left  the  plants  an  hour  in  a  .1^ 
solution  of  ammonia,  then  placed  them  for  12  hours  in  a 
\oi>  solution  of  ferrocyanide  containing  5^  of  acetic  acid,  then 
washed  in  cold  water,  and  finally  let  them  remain  for  12 
hours  in  a  not  too  dilute  solution  of  ferric  chloride.  Certain 
differentiations  of  the  cytoplasm  then  showed  an  evident 
blue  color. 


MICROCHEMIS TR  V.  13$ 

I.      Pepsin    and    Pancreatin. 

232.  Recently,  the  ferments  secreted  by  the  stomach  and 
pancreas,  which  have  the  power  of  converting  proteids  into 
soluble  compounds  (peptones),  have  been  used  for  their  mi-  • 
crochemical  recognition.  Both  ferments  can  now  be  obtained 
in  very  stable  form,  as  pepsin-glycerine  and  pancreatin-gly- 
cerine,  of  Dr.  G.  Griibler,  Leipzig. 

233.  Digestion  with  pepsin  maybe  accomplished  bykeep^ 
ing  the  objects  in  a  mixture  of  one  part  pepsin-glycerine 
and  three  parts  water,  acidified  with  .2^  of  its  weight  of 
chemically  pure  hydrochloric  acid,  for  an  hour,  at  a  temper- 
ature of  40°  C.  The  effect  of  hydrochloric  acid  may  be  ob- 
served in  control-experiments  containing  the  acid  alone. 

234.  Pancreatin-glycerine  maybe  diluted  with  three  times- 
its  volume  of  water  and  then  used  in  the  same  way. 

The  previous  treatment  of  the  objects  has  an  important 
influence  upon  the  reaction.  The  solution  of  the  proteids  takes 
place  most  easily  in  sections  taken  directly  from  the  living 
plant.  But,  in  general,  alcoholic  material  is  to  be  preferred, 
since  the  digestibility  of  the  substances  soluble  in  water  may 
thus  be  determined,  and  clearer  images  are  usually  obtained.. 
But  the  alcohol  should  act  for  as  short  a  time  as  possible  (24. 
hours),  since  it  may  influence  the  digestibility  by  its  longer 
action. 


d,     Nucleins. 

235.  Nucleins  have  been  prepared  especially  from  yeast,, 
from  the  thymus  gland  of  the  calf,  from  the  yolk  of  eggs, 
and  from  salmon-sperm.  These  are  distinguished  from  pro- 
teids especially  by  the  fact  that  they  contain  phosphorus.- 
In  other  respects  the  analyses  of  nucleins  from  differ- 
ent sources  show  considerable  differences.  Altmann  (II)  has 
lately  isolated  from  these  nucleins  bodies  of  uniform  compo- 
sition which  he  calls  nucleic  acids.  These  contain  about  9^ 
of  phosphorus  and  are  quite  free  from  sulphur  when  pure. 
They  are  precipitated  by  albumen,  and  Altmann  regards  the 


1 34  BO  TA  XICA  L   MICRO  TECHNIQ  UE. 

iiuclein  preparations  of  various  authors  as     compounds    of 
nucleic  acids  with  various  amounts  of  albumen. 

[Malfatti  (II)  has  prepared  an  artificial  nuclein  from  syn- 
tonin  and  metaphosphoric  acid,  which  yields  nucleic  acids 
when  treated  by  Altmann's  method.] 

236.  Zacharias  has  recently  (I  and  II)  attempted  to  recog- 
nize microchemically  the  general  distribution  of  nucleins, 
especially  in  cell-nuclei.  He  gives  as  a  characteristic  reac- 
tion for  them,  their,  insolubility  in  pepsin  and  hydrochloric 
acid  (cf.  §  233),  in  which,  as  well  as  in  .2-.3;^  h}'drochloric 
acid,  they  take  a  sharply  defined  and  peculiarly  glistening 
appearance.  But  the  nucleins  are,  according  to  Zacharias, 
soluble  in  a  \oi  solution  of  common  salt,  in  a  concentrated 
solution  of  sodium  carbonate,  in  dilute  caiistic  potasJi'^oXyiXAon, 
in  concentrated  hydrochloric  acid,  and  in  a  mixture  of  four 
parts  concentrated  hydrochloric  acid  with  three  parts  water. 
Further  investigations  must  show  how  far  the  macrochemi- 
cally  prepared  nucleins  and  those  recognized  by  the  above 
reactions  correspond  with  each  other. 

[According  to  Malfatti  (I)  and  Zacharias  (V),  the  nucleins 
seem  to  constitute  the  so-called  chromatin-bodies  of  the 
nucleus  (cf.  §  239).] 

c.  Plasiin 

237.  Reinke  (I)  prepared  a  nitrogenous  compound  from 
the  Plasmodia  of  ^thalinm  septicum^  which  he  calls  plas- 
tin.  According  to  Zacharias  (I  and  II),  this  compound  rep- 
resents the  fundamental  substance  of  the  cytoplasm  and 
agrees  in  its  microchemical  behavior  with  nuclein,  in  not 
being  attacked  hy  pepsin  with  hydrochloric  acid  and  in  being 
soluble  in  concentrated  hydrochloric  acid.  But  plastin  differs 
from  nuclein  in  not  swelling  in  a  10^  solution  of  salt  after 
treatment  with  pepsin,  and  in  being  less  readily  soluble  in 
alkalies  and  insoluble  in  a  mixture  of  four  parts  of  pure  con- 
centrated hydrochloric  acid  and  three  parts  water. 

It  remains  to  be  learned  whether  plastin  is  really  a  single 
compound  or  includes  a  group  of  related  compounds.     Ac- 


MICR  0  CHEMIS  TR  V.  135 

cording  to  Loevv  (I),  the  plastin  prepared  macrochemically 
byReinke  is  to  be  regarded  simply  as  an  impure  albuminoid 
preparation.  This  author  also  showed  that  the  cytoplasm 
regarded  by  other  authors  as  free  from  albumen  gives  the 
protein-reaction  after  being  first  treated  for  a  time  with  caustic 
potash  (cf.  §  228  and  Loew,  II). 


i/.     Cytoplasthi,    Chloroplastin,    Metaxin,    Pyrenin,    Amphi- 
pyrenin,  Chromatin^  Linin,  and  Paralinin. 

238.  According  to  the  investigations  of  Schwarz  (I),  the 
protoplasmic  body  is  made  up  of  eight  different  proteids 
which  are  limited  in  their  distribution  to  very  special  differ- 
entiations of  the  protoplasm,  and  which  should  be  compara- 
tively easy  to  distinguish  microchemically  according  to  the 
tables  of  their  reactions  prepared  by  this  author.  But  if  one 
examines  the  separate  results  of  his  observations,  as  described 
in  detail,  it  is  found  that  the  most  of  the  reagents  used  have 
given  very  different  results  even  with  the  few  objects  ex- 
amined, and  that  the  author's  own  observations  do  not  at  all 
always  correspond  with  the  special  statements  of  his  tables^ 
There  can  no  longer  be  any  doubt  that  the  substances  distin- 
guished by  Fr.  Schwarz  do  not  represent  uniform,  chemically 
definable  substances.  Further  studies  must  show  whether 
the  reagents  used  by  him  are  capable  of  rendering  good 
service  in  the  study  of  the  morphological  elements  of  the 
protoplasm.  This  seems  most  probable  in  case  of  the 
"  pretty  concentrated "  solution  of  copper  sulphate  used  by 
Schwarz  (I,  116),  which  dissolves  only  the  chromatin*  in  the 
nucleus  and  fixes  all  its  other  constituents  well.  A  mixture 
of  one  volume  of  a  10^  aqueous  solution  oi  pot  as  shun  ferro- 
cyanide,  two  volumes  of  water,  and  half  a  volume  of  glacial 
acetic  acid  also  dissolves  only  chromatin  ;  but  this  reagent 
is  not  adapted  to  fixing,  since  it  causes  swelling. 

239.  But,    since    the    nomenclature     introduced    by    Fr. 

*  [Malfatti  (I)  states  that  copper  sulphate  does  not  dissolve  chromatin,  but 
lorms  an  unstainable  precipitate  with  it.] 


136  BOTANICAL   MICROTECHNIQUE. 

Scliwarz  has  been  used  elsewhere  in  the  literature,  at  least 
the  names  of  his  eight  compounds  may  be  given  here. 

Two  of  them  occur  in  the  chlorophyll  granules,  chloro- 
plastin  and  metaxin.  The  first  of  these  represents  the  green 
iibrillse  within  the  chloroplasts,  and  between  them  is  the 
water-soluble  metaxin. 

In  the  nucleus  Schwarz  distinguished  five  substances, 
amphipyrenin,  which  forms  the  nuclear  membrane;  pyrenin, 
the  substance  of  the  nucleoli ;  chromatin,  the  strongly  stain- 
ing material  of  the  nuclear  framework ;  and  linin  and  para- 
]inin,  the  former  of  which  forms  a  fibrillar  network  in  the 
nucleus,  while  the  latter  fills  the  meshes  of  this  net. 

In  the  cytoplasm  Schwarz  finds  only  one  proteid,  which 
iie  calls  cytoplastin. 

13.  Ferments. 

240.  It  was  stated  by  Wiesner  (IV)  that  pepsin,  diastase, 
and  the  gum-ferment  described  by  him  give  characteristic 
color-reactions  with  orciyi  and  hydrochloric  acid,  which  are 
jnicrochemically  applicable.  But  Reinitzer  (II)  showed  that 
these  reactions  occur  with  various  carbohydrates,  and  most 
probably  depend  upon  the  fact  that  the  reagent  splits  off 
from  them  furfurol  or  related  compounds. 

But  Guignard  has  recently  tried  to  determine  the  location 
of  emulsin  and  myrosin,  partly  by  the  use  of  orcin. 

a.  Emulshi. 

240a.  Emulsin  splits  the  glucoside  amygdalifi,  contained 
in  bitter  almonds,  into  prussic  acid,  oil  of  bitter  almonds, 
and  sugar.  It  occurs,  according  to  Guignard  (III),  in  the 
leaves  of  Primus  Lauro-cerasus,  exclusively  in  a  parenchyma- 
tous sheath  surrounding  the  vascular  bundles.  This  author 
Teaches  this  conclusion  from  the  fact  that  only  these  cells 
form  prussic  acid  with  a  solution  of  amygdalin  ;  while  the 
spongy  and  palisade  parenchyma,  which,  on  the  other  hand, 
forms  prussic  acid  with  a  solution  of  emulsin,  is  plainly  to 
be  regarded  as  the  seat  of  amygdalin. 


MICROCHEMISTRY.  137 

The  contents  of  the  cells  containing  emulsin  become  red 
with  Millons  reagent,  and  violet  with  copper  sulphate  and 
caustic  potash.  It  seems  probable  that  these  reactions  are 
due  to  the  emulsin,  since  the  corresponding  cells  of  the 
emulsin-free  leaves  of  Cerasus  liisitanica  do  not  give  them. 

b.  Myrosin, 

241.  For  the  microchemical  recognition  of  myrosin,  which 
is  contained  in  many  Crticiferce,  Guignard  recommends  (II) 
concentrated  hydrochloric  rt:«<^  which  contains  a  drop  of  a 
loio  aqueous  solution  of  orci7i  in  each  ccm.  If  the  sections 
are  heated  in  this  solution  to  near  100°  C,  a  violet  color 
appears  in  the  cells  containing  myrosin. 

In  the  seeds  of  black  mustard  and  in  other  parts  of  various 
CrucifercB,  this  reaction  occurs  in  specialized  cells  rich  in 
proteids,  which  alone,  as  Guignard  has  experimentally  shown, 
are  able  to  decompose  potassium  myronate  (cf.  §  156). 

[Spatzier  (I)  finds  myrosin  also  in  the  Resedacece  and  in  the 
seeds  of  the  Violacece  and  Tropceolacece.  Where  it  occurs  in 
vegetative  organs  he  finds  it  in  a  dissolved  condition  ;  but 
in  dry  seeds  it  is  in  the  form  of  solid  homogeneous  granules 
of  about  the  size  of  aleurone  grains.] 


part  Z\)ivi>. 

METHODS  FOR  THE  INVESTIGATION  OF  THE 
CELL-WALL  AND  OF  THE  VARIOUS  CELL- 
CONTENTS. 

A.  The   Cell-wall. 

242.  Since  vegetable  cell-membranes  belong  to  the  class 
of  absorbent  bodies  and,  as  their  osmotic  relations  show, 
can  readily  give  passage  to  the  most  various  substances 
which  are  soluble  in  water,  it  may  be  assumed  that  they 
never  consist  simply  of  cellulose  and  water,  in  the  living 
plant.  Rather,  they  are  always  incrusted,  within  the  plants 
with  a  greater  or  less  amount  of  foreign  substances,  accord- 
ing to  their  age  and  position.  How  far  the  varying  chemi- 
cal and  physical  relations  of  vegetable  cell-membranes  are 
to  be  attributed  to  such  incrustations  of  organic  or  inorganic 
nature  cannot  at  present  be  certainly  determined. 

But  it  is  well  established  by  modern  researches  that  pure 
cellulose  is  much  less  than  the  other  constituents  in  many 
membranes  or  parts  of  membranes.  Indeed  it  is  very 
probable  that  cellulose  is  entirely  wanting  in  many  parts  of 
membranes. 

Positive  conclusions  concerning  these  questions  can  only 
be  reached  when  we  have  obtained,  by  macroscopic  re- 
searches, a  sure  means  of  distinguishing  the  different  con- 
stituents of  the  cell-wall.  But  our  knowledge  in  this  re- 
spect is  still  too  fragmentary  to  make  possible  any  uniform 

U3 


SPECIAL   METHODS.  I39 

system  of  the  constituents  of  the  cell-wall,  although  the 
macrochemical  study  of  the  cell-wall  has  been  taken  up 
in  various  aspects  in  recent  years. 

243.  I  prefer,  then,  to  discuss  first  in  the  following  pages 
the  kinds  of  membranes  which  may  primarily  be  distin- 
guished by  their  microchemical  relations.  For  some  time 
there  have  been  pretty  generally  distinguished  the  pure 
cellulose  wall,  the  lignified  wall,  the  cuticularized  or  su- 
berized  wall,  the  gelatinized  wall,  and  fungus-cellulose.  In 
connection  with  the  gelatinized  wall  may  be  discussed  the 
remaining  plant  mucilages  and  gums  and  the  jelly-formation 
of  the  Conjugates.  As  related  to  these  various  modifications 
of  the  membrane  may  be  mentioned  the  paragalactan-like 
substances  which  serve  as  reserve-materials,  callose,  and  the 
pectins.  Finally,  this  chapter  may  describe  the  preparation 
of  ash  and  siliceous  skeletons,  and  some  methods  which 
have  been  used  in  the  investigation  of  the  development  and 
finer  structure  of  the  cell-walls. 

I.  The  Cellulose  Wall. 

244.  Cellulose  is  a  carbohydrate  whose  empirical  compo- 
sition corresponds  to  the  formula  CeHjoOj.  It  is  especially 
characterized  by  its  solubility  in  ciiprammonia  and  in  con- 
centrated sulphuric  acid,  by  its  blue  or  violet  color  with 
iodine  and  sulphuric  acid  ox  with  chloroiodide  of  zinc,  and  by 
the  fact  that  from  its  hydrolytic  splitting  with  sulphuric 
acid  there  finally  results  a  fermentable  sugar  (glucose). 

245.  But  it  should  be  remarked  that  there  are  very  prob- 
ably different  substances  which  give  the  same  reactions,  and 
which  perhaps  represent  nearly  related  isomeric  compounds. 
Thus,  according  to  W.  HofTmeister  CI  and  II),  cellulose 
shows  very  varying  relations,  especially  toward  a  1-5^ 
caustic  soda  solution,  in  which  it  is  partly  soluble,  partly 
insoluble.  But  an  exact  microchemical  distinction  of  the 
kinds  of  cellulose  has  not  yet  been  possible,  and  it  is  there- 
fore best  for  the  present  to  call  all  membranes  or  parts  of 
membranes  which  show  the  above  reactions  pure  cellulose 


140  BOTANICAL   MICROTECHNIQUE. 

membranes,  any   ash    constituents   being,  of    course,  quite 
disregarded. 

246.  For  the  microchemical  recognition  of  the  cellulose 
membranes  the  following  reactions  are  used  : 

1.  Solubility  in  concentrated  sulpJiuric  acid.  This  begins 
with  a  strong  swelling,  which  finally  passes  into  complete 
solution. 

2.  Solubility  in  cupr ammonia.  This  reagent,  which  is  also 
known  as  Schweizers  reagent,  may  be  prepared  by  precipi- 
tating cupric  oxyhydrate  from  a  solution  of  cupric  sulphate 
with  a  dilute  solution  of  caustic  soda,  then  washing  the  pre- 
cipitate with  water  by  repeated  decantation  and  filtering, 
and  finally  dissolving  it  in  the  most  concentrated  ammonia- 
water. 

A  very  good  reagent  may  be  more  simply  prepared  by 
pouring  13-16^  ammonia-water  over  copper  turnings  and 
letting  the  whole  stand  in  an  open  bottle  (cf.  Behrens  II, 

55). 

Cuprammonia  can  be  preserved  for  only  a  limited  time. 

To  test   its  fitness   for  use,  one  may  use  cotton,  which  it 

should  completely  dissolve  at  once. 

3.  The  blue  color  with  iodine  and  sulphuric  acid.  Ac- 
cording to  Russow,  this  reaction  may  be  best  conducted  by 
treating  the  sections  first  with  an  aqueous  solution  of  \i> 
iodine  and  \\^  potassium  iodide,  and  then  adding  a  mixt- 
ure of  two  parts  concentrated  sulphuric  acid  and  one  part 
water. 

4.  The  violet  color  with  chloroiodide  of  zinc.  This  re- 
agent is  usually  prepared  by  dissolving  an  excess  of  zinc  in 
pure  hydrochloric  acid  and  then  evaporating  the  solution  to 
the  density  of  sulphuric  acid  in  the  presence  of  an  excess  of 
metallic  zinc  ;  the  solution  is  then  saturated  with  potassium 
iodide  and  finally  with  iodine.  The  chloroiodide  may  be 
more  simply  prepared  by  dissolving  25  parts  of  zinc  chloride 
and  8  parts  .of  potassium  iodide  in  8.5  parts  of  water,  and 
then  adding  as  much  iodine  as  will  dissolve  (Behrens  II,  54). 
[I  have  used  with  excellent  results  a  preparation  obtained 
by  dissolving  solid  commercial  chloroiodide  of  zinc,  a  moist 


SPECIAL   METHODS.  I4I 

Avhite  salt,  in  somewhat  less  than  its  own  weight  of  water, 
and  then  adding  sufficient  metallic  iodine  to  give  the  solu- 
tion a  deep  sherry-brown  color.  I  prefer  Griibler's  prepara- 
tion of  the  chloroiodide.]  These  solutions  remain  for  a  long 
time  unchanged,  especially  when  kept  in  the  dark.  The 
reaction  succeeds  best  when  the  sections  are  placed  directly 
in  the  concentrated  reagent. 

5.  Recently  a  number  of  reagents  containing  iodine  have 
been  recommended  by  Mangin  (VII),  which  act  in  the  same 
manner  as  chloroiodide  of  zinc  and  seem  to  be,  in  part, 
more  delicate  than  it. 

Of  these  reagents  I  have  used  with  good  results  a  calciiim- 
cJdoride-iodine  solution,  and,  instead  of  following  Mangin's 
somewhat  more  elaborate  method,  have  prepared  it  by 
adding  about  .5  gram  of  potassium  iodide  and  .1  gram  of 
iodine  to  lo  ccm.  of  a  concentrated  solution  of  calcium 
chloride  and  then,  after  gentle  warniing,  separating  the 
solution  from  the  excess  of  iodine  by  filtering  through 
glass-wool. 

This  solution,  in  which  the  sections  should  be  placed 
directly,  colors  lignified  membranes  yellow  to  yellow-brown, 
but  pure  cellulose  walls  become  first  rose-red  and,  after  a 
time,  violet.  According  to  Mangin,  it  should  be  kept  in 
the  dark. 

By  the  aid  of  iodine-phosphoric  acid  recommended  by 
Mangin,  one  obtains  a  very  deep  violet  coloring  of  cellulose 
walls,  while  lignified  and  suberized  walls  are  colored  yellow 
or  brown.  This  reagent  is  prepared  by  adding  a  small 
quantity  of  potassium  iodide  (about  .5  gram  to  25  ccm.)  and 
a  few  crystals  of  iodine  to  a  concentrated  aqueous  solution 
of  phosphoric  acid,  and  gently  warming  the  whole.  The 
sections  should  be  freed  of  all  water  adhering  to  their  sur- 
faces, by  means  of  filter-paper,  before  being  placed  in  this 
solution. 

Mangin  also  recommends  mixtures  of  aluminium  chloride 
or  stannic  chloride  with  iodine  and  potassium  iodide.  For 
the  manner  of  preparing  and  using  these  solutions,  Mangin's 
work  may  be  consulted. 


142  BOTANICAL   MICROTECHNIQUE. 

[Mangin  states  (VIII)  that  the  most  important  and  most 
characteristic  reaction  of  cellulose  is  its  conversion  into 
hydrocellulose  or  amyloid.  This  conversion  is  not  certainly 
accomplished  by  acids,  but  the  best  results  are  obtained  by 
treating  the  cellulose  with  a  saturated  alcoholic  solution  of 
sodium  or  potassium  hydroxide  and  then  transferring  it  to 
absolute  alcohol.  Cuprammonia  also  produces  the  same 
result.  The  above-described  reagents  for  cellulose  act 
promptly  with  hydrocellulose.] 

247.  Their  behavior  with  staining  media  can  also  be  used 
for  the  recognition  of  pure  cellulose  membranes.  These 
also  serve  in  delicate  sections,  microtome  sections  or  the 
like,  to  bring  out  better  the  network  of  cell-walls. 

HcBmatoxylin  is  especially  adapted  to  this  purpose,  Giltay 
(I)  having  first  observed  the  fact  that  it  stains  deeply  only 
the  unlignified  and  unsuberized  membranes.  It  may  be 
used  in  very  various  solutions  (e.g.  in  the  so-called  Bohmer's 
(cf.  §  315)  or  in  Delafield's  (cf.  §  314)  solution).  These  stain 
pure  cellulose  walls  deep  violet,  while  lignified  and  suberized 
membranes  remain  at  first  uncolored,  or  are  stained  yellow 
or  brown.  In  most  sections  an  exposure  of  a  few  minutes 
is  sufficient  for  a  deep  staining  of  the  membranes. 

248.  The  writer  has  used  haematoxylin  with  the  best 
results  for  the  recognition  of  the  closing  membrane  of  bor- 
dered pits  (Zimmermann  IV).  With  the  wood  of  Coniferce 
it  is  sufficient  to  leave  the  sections  for  fifteen  minutes  in 
Bohmer's  haematoxylin  solution  (cf.  §  315)  to  obtain  a  deep 
staining  of  the  "  tori  "  of  the  bordered  pits,  which,  naturally, 
come  out  most  sharply  after  clearing  in  Canada  balsam  (cf. 
§§  14-22). 

249.  Aniline  blue  and  methyl  blue  may  also  be  used  for 
staining  cellulose  walls.  These  produce  an  intense  stain  in 
an  hour  with  microtome  sections,  which  is  not  affected  by 
alcohol,  clove-oil,  or  xylol,  so  that  the  preparations  may  be 
mounted  in  Canada  balsam.  A  solution  of  Berlin  blue  acts 
in  the  same  way.  This  is  prepared  by  allowing  i  gram  of 
soluble  Berlin  blue  and  .25  gram  of  oxalic  acid  to  stand  sev- 
eral hours  with  a  little  distilled  water,  then  adding  100  ccm. 


SPECIAL   METHODS,  1 43 

of  water  and  filtering  (cf.  Strasburger  I,  622).  To  obtain  a 
sufficiently  deep  stain,  the  solution  must  usually  be  allowed 
to  act  for  several  hours.  It  is  not  washed  out  by  alcohol. 
[According  to  Mangin  (VIII),  pure  cellulose  is  readily 
stained  by  many  of  the  azo-colors,  as  by  orseillin  BB, 
crocein  and  naphtol  black  in  an  acid  solution,  or  by  Congo- 
red  and  benzo-purpurin  in  an  alkaline  solution.  Several  of 
the  dyes  recommended  heretofore  for  cellulose  walls  really 
stain  only  the  pectic  constituents  of  cell-membranes  (cf. 
§  292).  Such  are  methylene  blue,  aniline  brown,  and  chino- 
lin  blue.] 

250.  A  very  deep  and  permanent  staining  of  the  wall  is 
obtained,  according  to  Van  Tieghem  and  Douliot  (I),  by 
placing  sections,  after  all  cell-contents  have  been  removed 
by  eau  de  Javelle  and  caustic  potash,  and  after  thorough 
washing,  first  in  a  dilute  solution  of  tannin  for  one  to  two 
minutes  and  then,  as  quickly  as  possible,  in  a  very  dilute 
solution  oi  ferric  chloride.  They  are -at  once  removed  from 
the  latter  solution  and  enclosed  in  glycerine  or  Canada  bal- 
sam.    All  the  membranes  are  then  stained  a  deep  black. 

For  staining  the  younger  membranes  of  microtome  sec- 
tions, I  have  lately  found  Congo-red  well  adapted.  I  allow  it 
to  act  in  concentrated  aqueous  solution,  for  24  hours,  upon 
the  sections,  and  then  wash  them  in  alcohol  and  mount  in 
Canada  balsam. 

2.  Lignified  Membranes. 

251.  Lignified  membranes  are  distinguished  from  those 
of  pure  cellulose  by  being  insoluble  in  cuprammonia  and  by 
being  colored  yellow  or  brown  by  iodine  and  sulphuric  acid 
or  chloroiodide  of  zinc.  It  was  formerly  generally  believed 
that  this  difference  in  chemical  relations  of  lignified  walls 
is  due  to  the  incrustation  of  the  cellulose  with  a  substance 
richer  in  carbon,  lignin.  And  in  fact  lignified  membranes 
give  the  reactions  of  pure  cellulose  after  treatment  with 
Schulze's  macerating  mixture  (cf.  §  9).  According  to  Man- 
gin  (VII),  the  same  thing  occurs  after  treatment  with  eau  de 
Javelle. 


144  BOTANICAL   MICROTECHNIQUE. 

252.  Recently  the  attempt  has  been  made  in  several 
quarters  to  reach  more  accurate  conclusions  concerning  the 
chemical  composition  of  lignified  membranes,  and  especially 
as  to  the  constitution  of  lignin. 

Wholly  trustworthy  results  can,  of  course,  only  be  reached 
by  macrochemical  investigations  with  exact  quantitative  an- 
alysis. In  this  connection  should  be  mentioned  the  recent 
researches  of  Lange  (I  and  II),  who  has  isolated  from  the 
woods  of  the  beech,  oak,  and  fir,  two  compounds  of  an 
acid  character,  '^  lignic  acids,''  which  may,  however,  possibly 
come  from  a  single  substance.  Lange  also  obtained  various 
by-products  concerning  whose  significance  nothing  is  yet 
known. 

253.  There  is  also  widely  distributed  in  lignified  mem- 
branes a  gum-like  substance  which  Thomsen  has  called  wood- 
gum.  It  may  be  extracted  with  a  5^  solution  of  caustic  soda 
and  then  precipitated  from  this  solution  with  90^  alcohoL 
On  hydrolysis  wood-gum  yields  either  arabinose,  CgH^O^ ,. 
or  xylose,  C^H.^O,. 

Wood-gum  and  both  of  its  derivatives  above  mentioned 
take  a  cherry-red  color  on  warming  with  phloroglucin  and 
hydrochloric  acid.  But  Allen  (I,  39)  has  shown  that  the 
phloroglucin  reaction  about  to  be  described  is  not  to  be 
referred  to  the  wood-gum  ;  for,  on  one  hand,  the  reaction 
takes  place  with  lignified  membranes  in  the  cold,  and,  on 
the  other,  the  colors  which  appear  in  the  different  reactions 
show  very  different  spectroscopic  relations. 

254.  Attempts  have  also  been  made  to  determine  the 
chemical  constitution  of  lignified  membranes  bymicrochem- 
ical  studies.  Especially  Singer  (I)  and,  more  recently,  Heg- 
ler  (I)  have  tried  to  prove  that  coniferin  and  vanillin  always 
occur  in  lignified  walls.  This  view  is  based  chiefly  on  a 
series  of  color-reactions  which  lignified  walls  give  with  vari- 
ous aromatic  compounds.  I  give  a  compilation  of  the  chief 
of  these  reactions  with  remarks  on  their  application,  which 
should  receive  notice  here  because  the  reactions  may  be  used 
with  good  results  for  the  microchemical  recognition  of  lig- 
nification. 


SPECIAL   METHODS. 


145 


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146  BOTANICAL   MICROTECHNIQUE. 

255.  According  to  Hegler  (I,  40),  all  of  these  compounds 
that  have  been  tested  give  the  same  color-reactions  with  co- 
niferin  or  vanillin  or  with  a  mixture  of  both  substances  ;  and 
this  author  therefore  considers  it  demonstrated  that  both 
compounds  occur  constantly  in  lignified  membranes,  and  that 
they  are  the  cause  of  the  above  described  color-reactions. 

Of  the  above  enumerated  reagents  thallin  and  phenol  de- 
serve especial  attention,  since,  according  to  Hegler  (I),  the 
former  gives  the  described  color-reaction  only  with  vanillin, 
the  latter  only  with  coniferin."*  Since  thallin  colors  vanillin 
yellow,  but  phenol  colors  coniferin  blue,  bythe  use  of  a  mix- 
ture of  the  two  reagents  one  may  draw  certain  conclusions 
as  to  the  relative  abundance  of  the  two  substances,  accord- 
ing as  the  color  produced  is  yellower  or  bluer.  Hegler  (I,  58) 
has  u.sed  for  the  same  purpose  a  solution  prepared  by  mixing 
.5  gram  of  thallin  sulphate,  1.3  grams  of  thymol,  2  ccm.  of 
water,  26.5  ccm.  of  alcohol,  and  .5  gram  of  potassium  chlorate, 
and  diluted  for  use  with  its  own  volume  of  hydrochloric  acid 
of  specific  gravity  1.124.  This  author  draws  from  his  studies 
carried  on  with  this  reagent,  the  conclusion  that  the  younger 
xylem-cells  contain  more  coniferin  than  vanillin,  but  that 
the  older  ones  are  rich  in  vanillin  and  less  so  in  coniferin. 

256.  On  the  other  hand,  it  is  to  be  noticed  that,  besides 
vanillin  and  coniferin,  other  substances  containing  the  alde- 
hyde group  give  with  the  compounds  mentioned  identical  or 
similar  color-reactions  to  those  of  Hgnified  cell-walls  ;  accord- 
ing to  Ihl  (I),  cinnamic  aldehyde,  and,  according  to  Nickel, 
salicylic  aldehyde.  It  may  therefore  be  considered  as  good 
as  proven  that  the  color-reactions  of  lignified  membranes 
depend  upon  the  presence  of  one  or  of  various  compounds 
belonging  to  the  aldehyde  group.  Seliwanoff 's  observations 
also  support  this  view.  According  to  these,  lignified  walls 
are,  on  one  hand,  colored  red  by  a  solution  of  fuchsin  decol- 
orized with  sulphurous  acid,  and,  on  the  other  hand,  no 
longer  give  the  reactions  of  lignified  membraties  with  phloro- 


♦The  correctness  of  Hegler's  statement  that   thymol  also  gives  no  color 
ivith  vanillin  has  lately  been  disputed  by  Molisch  (I,  48,  Note  3). 


SPECIAL    METHODS.  14/ 

glucin  etc.  after  treatment  with  hydroxylamine,  which 
chemically  unites  with  aldehydes,  destroying  the  aldehyde 
group  (cf.  Nickel  II,  755). 

In  what  relation  these  aldehyde-like  compounds  stand  to 
the  so-called  lignin  cannot  at  present  be  stated. 

257.  For  the  microchemical  recognition  of  lignification, 
besides  the  behavior  with  cuprammonia  and  with  iodine  solu- 
tion already  described  (§  251),  the  color-reactions  given  in  the 
foregoing  table  may  be  used.  Of  these  reagents,  aniline 
sulpJiate  and  phloroghicin  are  especially  good.  The  colors 
produced  by  these  substances  last  but  a  short  time,  while 
tJiallin  gives  permanent  colors  and  is  therefore  adapted  to 
the  making  of  permanent  preparations,  which  may  be 
mounted  in  glycerine-gelatine  or  in  Canada  balsam. 

258.  Various  pigments  may  also  render  good  service  in 
the  study  of  lignified  walls.  These  behave  quite  differently 
from  unlignified  walls  with  staining  media  and  therefore  cer- 
tain staining  solutions  maybe  well  used  for  the  distinction  of 
the  different  sorts  of  membranes. 

259.  For  the  staining  of  lignified  membranes  fuchsifi  has 
shown  itself  especially  useful,  and  has  already  been  recom- 
mended by  Van  Tieghem  and  Berthold  for  this  purpose.  I 
•obtained  very  beautiful  permanent  preparations,  in  which 
only  the  lignified  walls  were  stained  deep  red,  by  leaving  the 
microtome  sections  first  for  a  quarter  of  an  hour  or  longer  in 
an  aqueous  solution  of  fuchsin,  and  then  washing  them  for  a 
short  time  in  a  solution  of  picric  acid,  such  as  Altmann's, 
which  contains  one  part  of  a  concentrated  alcoholic  solution 
of  picric  acid  to  two  parts  of  water  (cf.  §  345).  This  makes 
them  dark-colored,  and  much  of  the  color  is  then  washed 
out  with  alcohol ;  finally  they  are  passed  into  xylol  and 
xylol-Canada  balsam. 

If  a  double  staining  is  desired,  it  may  be  obtained  by  plac- 
ing the  sections,  after  the  washing  in  alcohol,  for  an  hour  in 
a  suitable  solution  of  haematoxylin,  such  as  Bohmer's  (cf.  § 
315),  aniline  blue,  methyl  blue,  or  Berlin  blue.  After  wash- 
ing  again  in  alcohol  and  mounting  in  Canada  balsam,  one 
obtains  preparations  in  which   the  lignified  membranes   are 


148  BOTANICAL   MICROTECHNIQUE, 

stained  deep  red    and  pure  cellulose  membranes  violet  or 

blue. 

260.  Acid  fiichsin*  gave  me  similar  preparations,  which 
were  washed  either  with  running  water  or  with  Altmann's 
picric  acid  solution  (cf.  §§  345-6)-  Aniline-water-j^/r^wz;? 
also  gave  me  preparations  in  which  only  the  lignified  and 
suberized  walls  were  stained  after  being  washed  out  with 
acid  alcohol  (cf.  §  268),  after  acting  for  at  least  an  hour. 
Gentian  violet  acted  in  the  same  way  when  used  according 
to  Gram's  method  (§321).  All  these  preparations  maybe 
well  preserved  in  Canada  balsam,  and  the  dyes  may  be  used 
for  double  staining  in  the  way  above  described. 

261.  The  staining  methods  named  may  be  very  well  used 
for  the  demonstration  of  the  course  of  the  vascular  bundles 
in  whole  parts  of  plants,  such  as  leaves  or  thin  stems.  I 
obtained  very  instructive  preparations  with  a  concentrated 
aqueous  solution  of  fuchsin  under  which  I  cut  off  the  part 
to  be  stained,  so  that  the  vascular  bundles  were  mostly 
deeply  stained  in  a  relatively  short  time.  When  this  was 
accomplished,  I  placed  pieces  of  the  objects  in  alcohol  until 
the  chlorophyll  was  completely  removed,  cleared  them  in 
clove-oil,  and  transferred  to  Canada  balsam.  Especially 
favorable  objects  for  study  are  found  in  the  leaves  of  Secale 
cereale  and  of  Impatiens  parviflora.  In  the  latter  only  the 
tracheal  elements  were  colored  red,  and  could  be  very 
clearly  seen  even  in  the  thicker  parts. 

3.  The  Cuticle  and  Suberized  Membranes. 

262.  Until  recently  it  has  been  generally  assumed  that 
suberization  is  due  to  the  incrustation  of  the  cellulose  wall 
with  a  fat-like  substance  commonly  called  suberin.  This 
assumption  has  been  based  especially  on  the  observation 
of  Fr.  von  Hohnel  that  suberized  membranes  are  colored 
red-violet  with  chloroiodide  of  zinc  after  treatment  with 
an    aqueous    solution    of   caustic    potash.      But   the    recent 

*  This  dye  is  also  called  "  Fuchsin  S,"  and  by  Dr.  Grtibler,  "  Fuchsin  S 
after  Weigert." 


SPECIAL    METHODS.  1 49 

researches  of  Gilson  (I)  have  made  it  at  least  very  question^ 
able  if  there  is  any  cellulose  present  in  suberized  walls. 
This  author  isolated  from  the  cork  of  various  plants  an  acid,. 
phellonic  acid,  which,  as  well  as  its  potassium  salt,  becomes 
rose-  or  copper-red  with  chloroiodide  of  zinc.  Gilson  be- 
lieves that  the  cause  of  the  violet  or  rather  reddish  color 
which  these  membranes  treated  with  caustic  potash  assume 
with  chloroiodide  of  zinc  is  to  be  sought  in  the  presence  of 
potassium  phellonate.  In  fact,  the  staining  described  does 
not  appear  if  the  walls  are  extracted  with  boiling  alcohol 
after  treatment  with  caustic  potash,  and  before  the  addition 
of  the  chloroiodide.  The  color  which  appears  after  prelimi- 
nary treatment  with  chromic  acid  is  probably  due,  according 
to  Gilson,  to  the  formation  of  free  phellonic  acid.  The 
absence  of  color  after  treatment  with  cuprammonia  is  due,, 
not  to  the  solution  of  cellulose,  but  to  the  conversion  of 
potassium  phellonate  into  the  copper  salt,  which  takes  a 
yellowish-brown,  very  slightly  characteristic  color  with  chlo- 
roiodide of  zinc.  Finally,  the  presence  of  cellulose  in  the 
suberized  wall  is  rendered  improbable  by  the  fact  that  the 
whole  suberin  lamella  may  be  made  to  disappear,  according 
to  Gilson,  by  long  continued  treatment  with  a  3^  boiling* 
alcoholic  solution  of  potassium  hydrate,  which  does  not 
recognizably  attack  cellulose. 

263.  Besides  phellonic  acid,  already  described,  to  which 
Gilson  assigns  the  formula  C^H^Og,  two  other  acids  have 
been  isolated  from  the  cork  of  Querciis  Suber  by  the  same 
author,  suberic  acid  (Ci^HjoOg)  diud  phloionic  acid  {C^.H^^O J). 
It  still  remains  undetermined  in  what  form  these  acids  are 
contained  in  suberized  membranes.  But  it  is  not  probable 
that  they  occur  as  true  glycerine  ethers,  since  the  suberin 
lamella  is  insoluble  in  all  solvents  for  fats  and  could  not  be 
melted  by  Gilson  when  heated  up  to  290°  C.  The  view  sug- 
gested by  Kiigeler  (I,  44)  that  suberin  is  so  difficult  of  solu- 
tion because  the  suberin  molecules  are  enclosed  between 
cellulose  molecules  is  untenable,  since  it  is  shown  that  the 
suberin  lamella  contains,  at  most,  only  traces  of  cellulose. 
Therefore,  at   present,  Gilson's   view   that   suberin   consists 


150  BOTANICAL   MICROTECHNIQUE. 

of  compound  ethers  or  of  condensation-  or  polymerization- 
products  of  various  acids  seems  to  have  most  in  its  favor. 

[Van  Wissehngh  has  lately  (I)  found  that  most  of  the  con- 
•stituents  of  suberin  melt  at  a  temperature  below  ioo°  C, 
but  are  deposited  in  a  substance  that  does  not  melt  and 
must  first  be  removed.  He  finds  that  the  suberin  constitu- 
ents are  mostly  soluble  in  chloroform,  and  believes  that  they 
<onsist  of  various  fatty  substances  with  glyceril  or  other 
<:ompound  ethers.] 

264.  In  this  connection  the  optical  relations  of  suberized 
walls  are  worthy  of  attention,  since  they  enable  us  to  draw 
some  conclusions  as  to  the  form  in  which  the  substances  in 
<luestion  occur  in  the  membranes.  The  suberized  mem- 
branes, as  well  as  the  cuticle,  show  a  pretty  strong  double 
refraction,  and  their  optical  axes  are  usually  placed  in  the 
reverse  position  to  those  of  the  pure  cellulose  wall.  But 
this  double  refraction  disappears  completely,  as  Ambronn 
has  shown  (I),  on  heating  to  100°  C,  reappearing  as  before 
on  cooling.  This  may  be  easily  seen  by  heating  cross- 
sections  of  the  leaf  of  Agave  americana  in  glycerine  until 
the  fluid  boils  and  then  examining  them  with  a  polarizing 
microscope. 

The  optical  relations  of  cork  clearly  compel  the  view  that 
its  double  refraction  is  due  to  the  presence  of  regularly 
arranged  particles  of  crystalline  form,  which  melt  on  heating 
and,  on  subsequent  cooling,  recrystallize  in  the  same  regular 
arrangement. 

265.  It  remains  to  be  determined  by  further  studies 
whether  all  suberized  membranes  have  the  same  composi- 
tion, and  to  what  extent  the  external  layers  of  the  epider- 
mal cells,  the  cuticle  and  the  cuticular  layers,  agree  in 
material  constitution  with  the  suberin  lamella  of  cork  cells. 

266.  As  has  been  long  known,  suberized  membranes,  as 
"well  as  cuticularized  ones,  show  the  following  relations  to 
chemical  reagents  : 

They  are  insoluble  in  cuprammonia,  are  never  colored 
blue  or  violet  by  iodine  and  sulphuric   acid  or  by  chloro- 


SPECIAL   METHODS.  15E 

Iodide  of  zinc,  but  always  yellow  or  brown  ;  and  are  in- 
soluble in  concentrated  sulphuric  acid. 

But,  according  to  Fr.  von  Hohnel's  researches  (I),  the  fol-^ 
lowing  reactions  are  especially  characteristic  : 

Concentrated  caustic  potash  solution  causes  in  the  cold  a 
yellow  coloring  of  suberized  membranes,  which  increases  ia 
intensity  when  they  are  warmed  in  this  fluid.  The  suberized 
membranes  take,  at  the  same  time,  a  lineate  or  granular 
structure  which  becomes  plainer  on  further  warming.  Ort 
boiling  in  the  same  solution,  the  large  yellow  drops  which 
are  formed  often  escape  entirely  from  the  membrane. 

Schulzes  jnacerating  mixture  (cf.  §  9,  i)  is  resisted  longest 
by  suberized  walls,  of  all  the  modifications  of  cellulose;  but 
they  finally  run  together,  on  long  boiling  in  the  fluid,  into 
oil-like  drops  whose  substance  is  termed  eerie  acid  and  is  sol- 
uble in  hot  alcohol,  ether,  chloroform,  benzol,  and  a  dilute 
solution  of  caustic  potash,  but  insoluble  in  carbon  bisul- 
phide. 

Concentrated  chromic  acid  either  does  not  dissolve  the 
suberized  membranes  at  all,  or  only  after  acting  for  a  day;; 
while  all  the  other  modifications  of  cellulose,  except  fungus- 
cellulose,  are  dissolved  by  this  acid  in  a  short  time. 

267.  For  distinguishing  between  suberized  and  lignified 
cell-membranes,  chlorophyll  and  alcannin  may  be  used. 

Correns  (II,  658,  note)  first  recognized  the  fact  that  ck/o- 
rophyll  stains  the  cuticle  and  suberized  membranes  deep 
green,  while  the  lignified  and  pure  cellulose  walls  are  not 
colored.  For  this  purpose,  a  freshly  prepared  alcoholic 
solution  of  chlorophyll,  as  concentrated  as  possible,  should 
be  allowed  to  act  on  the  sections  in  darkness  for  a  quar- 
ter of  an  hour  or  longer.  The  sections  may  then  be  ex- 
amined in  water.  They  cannot  be  preserved  by  ordinary 
methods. 

For  staining  with  alcannin^  a  solution  of  this  substance  in 
50^  alcohol  is  used,  in  which  the  sections  are  left  for  several 
hours  or  longer.  All  suberized  membranes  and  the  cuticle 
take  a  red  color  which  is  not  so  deep  as  with  any  fats  which 
may  be  present,  but  is  always  clearly  visible. 


152  BOTANICAL   MICROTECHNIQUE. 

Both  of  these  stains  are  of  interest  as  indicating  anew 
that  fat-Hkc  substances  are  deposited  in  the  cuticle  and  cork. 

267a.  I  have  recently  found  that,  besides  alcannin,  which 
gives  a  very  deep  staining  of  the  cuticle  after  long  action, 
/>smic  acid  2iV\A  cyanin  maybe  also  used  for  the  recognition 
of  suberized  membranes.  For  this  purpose,  I  dissolve  cya- 
nin  in  50^  alcohol  and  add  an  equal  volume  of  glycerine. 
Preliminary  treatment  of  sections  with  eau  de  Javelle  is  gen- 
erally to  be  recommended,  as  it  destroys  the  tannins  which 
hinder  the  staining.  It  also  causes  the  lignified  walls  to  lose 
their  power  of  staining,  while  suberized  ones  are  as  deeply 
stained  as  in  the  fresh  condition,  even  after  being  exposed 
for  a  day  to  its  action  (cf.  Zimmermann  VII). 

268.  In  their  behavior  with  staining  media,  the  cuticular- 
ized  and  suberized  membranes  show  in  many  respects  an 
agreement  with  lignified  ones.  This  is  especially  true  of  the 
•so-called  suberin  lamella  of  cork-cells;  but  the  true  cuticle 
is  often  less  easily  stainable.  But  it  is  usually  easy  to  stain 
the  cuticular  layers  differentially,  especially  in  thick-walled 
epidermal  cells  ;  and  double  stainings,  in  which  these  layers 
are  differently  colored  from  the  cellulose  layers  lying  be- 
neath, may  be  obtained.  But  it  must  be  remarked  that  these 
stains  do  not  always  act  with  the  same  precision,  in  all  cases, 
as  with  lignified  walls ;  and  different  plants  do  not  appear  to 
behave  in  the  same  way  in  this  respect.  I  recommend  as 
suitable  objects  for  study,  the  leaves  of  Clivia  nobilis  or  Agave 
americana.  On  these  the  following  stainings  and  double 
stainings  may  be  readily  carried  out.  The  statements  as  to 
time  refer  to  microtome  sections. 

a.     Safranin. 

269.  The  best  staining  medium  for  suberized  walls  is  ani- 
line-water-safranin,  prepared  by  mixing  equal  volumes  of  ani- 
line-water and  a  concentrated  alcoholic  solution  of  safranin. 
I  allow  this  to  act  for  half  an  hour  or  longer  on  the  sections, 
then  cover  them  with  acid  alcohol,*  which  is  quickly  replaced 


*  Thai  Is,  alcohol  to  which  is  added  about  .55^  of  the  ordinary  chemically 
pure  HCI. 


SPECIAL   METHODS.  153 

by  alcohol.  They  are  washed  with  the  latter  until  no  more 
color  is  given  off,  and  then  transferred  to  Canada  balsam  in 
the  usual  way.  Especially  if  the  washing  with  acid  alcohol 
is  just  right,  only  the  lignified  and  suberized  cell-walls  are 
stained  in  these  preparations,  in  which  the  former  show  a 
bluish,  the  latter  a  rather  yellowish,  color. 

If  it  is  desired  to  stain  the  cellulose  walls  also,  this  maybe 
done  by  one  of  the  following  methods  : 

a.     Methyl    Blue. 

The  sections,  stained  with  safranin  and  washed  with  alco- 
hol, are  placed  in  a  concentrated  aqueous  solution  of  methyl 
blue,  in  which  they  remain  a  quarter  of  an  hour  or  longer. 
They  are  then  washed  in  alcohol  and  mounted  in  Canada 
balsam.  The  cellulose  walls  are  then  stained  blue,  the  su- 
berized and  lignified  ones,  red. 

/?.     Aniline    Blue 

This  must  be  used  in  aqueous  solution  which  must  be 
first  washed  off  with  water  after  the  staining,  since  turbidity 
readily  results  from  the  direct  addition  of  alcohol.  Other- 
wise it  is  used  like  methyl  blue. 

y.     Haematoxylin.  , 

Bohmer's  haematoxylin  (§  315)  may  well  be  used  for  double 
staining  with  safranin.  This  is  allowed  to  act  for  a  few  min- 
utes on  sections  stained  with  safranin  and  washed,  is  then 
washed  off  with  water,  and  the  sections  are  mounted  in 
Canada  balsam.  Sections  thus  treated  show  the  lignified 
and  suberized  walls  red,  and  the  cellulose  walls  violet. 

b.     Gentian  Violet  and  Eosin. 

270.  In  the  so-called  Gram's  staining  process  (§  321)  with 
gentian  violet,  only  the  lignified  and  suberized  walls  remain 
stained  after  thorough  washing  with  clove-oil  ;  but  a  fine 
double  staining  may  be  obtained  by  proceeding  according  to 
Gram's  method  and  adding  to  the  clove-oil  used   in  washing 


154  BOTANICAL   MICROTECHNIQUE. 

a  little  eosin,  which  dissolves  readily  in  it.  The  eosin  at 
once  stains  the  cellulose  walls  a  beautiful  red,  while  not 
changing  the  staining  of  the  other  walls. 

c.  Ammonia-fuchsin. 

271.  As  was  first  recognized  by  Van  Tieghem,  ammonia- 
fuchsin  is  well  adapted  to  staining  suberized  and  lignified 
membranes.  It  is  prepared  by  adding  ammonia  to  a  not 
too  concentrated  alcoholic  solution  of  fuchsin,  until  the  solu- 
tion becomes  straw-yellow  after  a  little  shaking.  The  solu- 
tion should  be  filtered  after  a  few  days,  but  can  be  kept  only 
a  few  weeks,  even  in  well  closed  bottles. 

A  double  staining  may  be  had  by  placing  the  sections 
first  in  the  above  described  ammonia-fuchsin  solution  for  a 
few  minutes,  and  then  passing  them  directly  to  an  aqueous 
solution  of  methyl  blue,  in  which  they  are  left  a  quarter  of 
an  hour  or  longer,  then  washing  with  alcohol  and  mounting 
in  Canada  balsam. 

d.  Cyanin  and  Eosin. 

272.  If  sections  are  placed  for  several  hours  in  a  freshly 
prepared,  very  dilute  aqueous  solution  of  cyanin,  which  may 
be  prepared  by  adding  20  drops  of  a  concentrated  alcoholic 
solution  to  100  ccm.  of  water,  the  lignified  and  suberized 
membranes  appear  beautifully  blue  after  washing  in  alcohol. 
If  clove-oil  containing  eosin  be  used  in  transferring  to  Can- 
ada balsam,  a  fine  double  staining  is  obtained.  The  modified 
walls  are  blue,  the  cellulose  walls  red. 

I  obtained  also  a  deep  staining  of  the  cuticle  by  leaving 
sections  for  a  considerable  time  in  a  solution  of  cyanin  in 
50j^  alcohol  and  then  washing  out  the  .stain  with  glycerine. 

4.    Gelatinized  Cell-walls,  Plant-mucilages,  and  Gums. 

273.  The  so-called  gelatinized  membranes  are  distin- 
guished from  cellulose  walls  chiefly  by  their  different  physi- 
cal character,  their  strong  power  of  swelling;  and  indeed 
there  occur  all  stages  between  pure  cellulose,  which  takes  up 
little  water,  and  the  gums  which  are  wholly  soluble  in  water, 


SPECIAL    METHODS.  I  55 

like  gum  arable.  Part  of  these  substances  are  formed  from 
cellulose,  but  most  of  them  are  formed  by  the  plant  directly 
as  mucilages.  It  should  be  observed  that  the  occurrence  of 
vegetable  mucilages  and  gums  within  the  plant  is  not  at  all 
restricted  to  the  cell-wall,  but  they  may  also  be  formed 
within  the  protoplasm.  But  it  has  seemed  to  me  best  to 
discuss  all  these  bodies  together  here,  in  view  of  their  un- 
doubted relationships,  which  may  lead  one,  with  Beilstein 
(I,  877),  to  group  them  under  the  designation^?/;;/^. 

274.  So  far  as  the  chemical  relations  of  the  gums  are  con- 
cerned, it  should  first  be  observed  that  most  of  them,  so  far 
as  they  have  been  analyzed,  agree  in  their  percentage  com- 
position with  cellulose  and  thus  correspond  to  the  formula 
CgHjoO^.  But,  on  the  other  hand,  they  differ  considerably 
from  cellulose  in  their  chemical  relations,  and  also  show^ 
great  differences  among  themselves. 

Thus  some  of  them  are  colored  blue  by  iodine  alone,, 
others  only  by  iodine  and  sulphuric  acid  or  chloro'iodide  of 
zinc,  and  still  others  are  colored  only  yellow  or  not  at  all  by 
iodine  preparations. 

A  part  of  the  gums  are  soluble,  a  part  quite  insoluble,  in. 
c'lipranunonia. 

On  oxidation  with  nitric  acid,  a  part  of  them  give  oxalic 
acid,  (COOH),,  a  part,  mucic  acid,  (CHOHX.(COOH}^,  a. 
part,  both  acids. 

Unfortunately  the  chemical  characters  of  the  various 
gums  are  not  determined  with  sufficient  exactness  to  make 
possible  a  strictly  scientific  grouping  of  them.  But  in  the 
following  account  some  remarks  on  the  general  methods  o£' 
recognizing  the  gums  may  be  in  place,  and  then  the  chief 
chemical  characters,  and  especially  the  microchemically 
applicable  reactions,  of  the  gums  which  have  been  studied 
in  detail  may  be  brought  together. 

275.  For  the  microchemical  recognition  of  the  gums  their 
strong  power  of  szvclling  in  water  may  first  be  used.  To 
follow  the  process  of  swelling  exactly  with  the  microscope, 
one  may  first  place  the  objects  in  absolute  alcohol,  in  which 
all    the    gums    are    insoluble   and    do    not    swell,  and    then 


156  BOTANICAL  MICROTECHNIQUE. 

gradually  allow  water  to  enter  from  the  edge  of  the  cover- 
glass. 

The  dissimilar  behavior  of  the  gums  with  iodine  solutions 
and  with  cuprammonia  has  already  been  mentioned.  Be- 
sides these,  corallin  may  be  used  in  many  cases  in  the  study 
of  gelatinized  cell-walls  and  gums,  since  many  of  them  are 
deeply  stained  by  it.  Since  it  is  practically  insoluble  in 
water,  it  may  be  dissolved  in  a  concentrated  solution  of 
soda.  This  solution  gradually  decomposes,  but  preserves 
its  staining  power  for  a  long  time  (cf.  also  §  289). 

Characteristic  stainings  of  plant-mucilages  are  often  ob- 
tained with  Hanstein's  aniline  mixture.* 

a.  Amyloid. 

276.  The  substance  known  by  the  name  amyloid  occurs 
in  the  seeds  of  various  plants  {TropcBoltnn  majus,  Impatiens 
Balsavtina,  PcBonia  officinalis,  many  Prirnulacece,  and  others) 
and  constitutes  a  reserve  material  which  goes  into  solution 
on  the  germination  of  the  seed. 

Amyloid  is  characterized  by  being  colored  blue  by  iodine 
solutions,  the  best  adapted  for  this  reaction  being,  according 
to  Nadelmann  (I,  616),  a  dilute  solution  of  iodine  and 
potassium  iodide,  since  a  concentrated  solution  of  the  same 
substances  colors  it  brownish  orange,  and  fresh  tincture  of 
iodine  does  not  generally  color  it  at  all  at  first. 

In  cuprammonia  amyloid  is  insoluble. 

Its  behavior  with  nitric  acid  is  also  characteristic. 

In  an  acid  which  contains  30^  of  HNO3  (spec,  gravity 
1.285),  amyloid  at  once  swells  strongly,  and  after  a  time 
becomes  entirely  dissolved  (cf.  Reiss  I,  735,  737,  739). 

The  amyloid  contained  in  the  seeds  named  is  not  identical 
with  the  compound  prepared  from  cellulose  by  treatment 
with  acids  (§  246),  which  has  often  been  termed  amyloid 
(cf.  Beilstein  I,  863,  882).  Amyloid  is  distinguished  from 
reserve-cellulose  (cf.   §   286)    by  the    reactions   already  de- 


*  [This  consists  of  an    alcoholic  solution  of   equal    parts  of    fuchsin  and 
methyl  violet.,] 


SPECIAL    METHODS.  15/ 

scribed  and  by  the  fact  that  its  hydrolytic  spHtting  with 
sulphuric  acid  yields  no  seminose,  but  most  probably  glu- 
cose (cf.  Reiss  I,  761). 

[Winterstein's  (I)  recent  researches  give  results  which 
differ  in  several  respects  from  those  of  Reiss.  He  finds 
amyloid  soluble  in  cuprainmonia  after  a  day.  From  this 
solution  it  is  not  precipitated  by  acids,  but  is  thrown  down 
by  alcohol.  Its  composition  seems  to  correspond  to  the 
formula  C^HgoOj^,  and  it  appears  to  belong  to  Tollens'  group 
of  Saccharo-colloids,  though  it  is  not  certain  that  it  is  a  single 
compound.  In  spite  of  its  bluing  with  iodine,  it  cannot  be 
regarded  as  very  nearly  related  to  starch.] 

b.  Wound-gum. 

277.  The  name  wound-gum  is  commonly  given  to  a 
substance  which,  according  to  Temme's  researches  (I),  is 
very  abundantly  secreted  in  the  vessels  by  the  surround- 
ing starch-cells,  in  natural  and  artificial  wounds,  and,  like 
tyloses,  closes  their  cavities.  This  wound-gum  agrees,  ac- 
cording to  Temme,  with  many  sorts  of  gums  in  that  it 
yields  oxalic  and  mucic  acid  on  oxidation  with  nitric  acid. 
But  it  differs  essentially  from  all  gums  in  not  swelling  in 
water  and  in  being  insoluble  even  in  caustic  potash  and  sul- 
phuric acid.  As  has  been  recognized  by  Temme,  wound- 
gum  is  stained  deep  red  by  pJiloroglucin  and  hydrochloric 
acid.  Molisch  showed  later  (IV,  290)  that  it  behaves  just 
like  lignified  membranes  with  aniline  sulphate,  metadiainido- 
henzol,  orcin,  and  thymol ;  and  he  believes  that  wound-gum 
contains  vanillin  in  solution  (cf.  §  254). 

c.  The  Gelatinous  Sheaths  of  the  Conjugatae, 

278.  In  many  Zygnemacece  the  whole  surface  of  the  cell- 
filaments  is  surrounded  by  a  colorless  covering,  a  ''gelatin- 
ous sheath,"  while  in  the  DesmidiacecB  the  excretion  of  jelly 
is  often  limited  to  distinct  regions  on  the  membrane  (cf. 
Klebs  II,  and  Hauptfleisch  I). 

Since  the  refractive  index  of  these  jelly-sheaths  differs 
but  little  from  that  of  water,  they  can  be  well  recognized. 


158  BOTANICAL   MICROTECHNIQUE. 

when  unstained,  only  by  the  aid  of  strong  objectives.  But 
with  lower  powers  they  stand  out  clearly  when  the  algae 
are  placed  in  very  finely  rubbed  India  ink,  according  to 
the  method  proposed  by  Errera  (III).  For  this  purpose, 
enough  of  the  genuine  Chinese  '*  India  ink"  may  be  rubbed 
up  directly  on  the  slide  to  give  the  drop  a  dark-gray  appear- 
ance, and  then  the  alga  to  be  studied  is  placed  in  it. 

No  trustworthy  statements  can  yet  be  made  as  to  the 
chemical  composition  of  these  jelly-masses  ;  and  it  need 
only  be  said  that  they  give  no  cellulose  reactions  either 
with  iodine  and  sulphuric  acid  or  with  chloroiodide  of  zinc, 
and  that  they  are  always  sharply  defined  against  the  cellu- 
lose wall  and  are  not  in  genetic  connection  with  it. 

A  number  of  observations,  made  especially  by  Klebs  (II) 
on  the  gelatinous  sheaths  of  the  Zygnernacece,  deserve  more 
detailed  notice,  as  they  show  that  these  must  possess  a  very 
complicated  organization. 

279.  Klebs  first  established  the  fact  that  the  gelatinous 
sheaths  always  consist  of  two  different  substances,  one  of 
which  can  be  extracted  with  hot  water  and  is  pretty  deeply 
stained  by  certain  dyes,  like  methylene  blue,  methyl  violet, 
and  vesuvin  ;  while  the  substance  which  is  insoluble  in  hot 
water  remains  quite  colorless  with  these  stains.  After 
staining  with  one  of  the  colors  above  named,  delicate  rods 
are  seen  in  the  sheaths,  which  often  appear  united  into  a 
fine  network  at  the  ends  which  are  directed  toward  the 
cell-lumen  (cf.  Fig.  33,  /).  The  same  structure  can  also  be 
made  visible  by  other  means,  especially  by  alcohol.  It  is 
evidently  due  to  the  fact  that  the  different  substances  are 
unequally  distributed  in  the  jelly-sheath. 

280.  A  further  remarkable  character  of  the  jelly-sheaths 
consists  in  the  fact  that,  after  the  deposition  in  them  of 
certain  precipitates,  for  instance,  of  Berlin  blue,  these  are 
thrown  out,  together  with  a  greater  or  less  part  of  the 
water-soluble  substances  of  the  sheath,  with  swelling  of  the 
latter  (cf.  Fig.  33,  //  and  ///).  This  "  throwing  off  of  the 
gelatinous  sheath"  begins  with  an  accumulation  of  the  pre- 
viously evenly  scattered  particles  into  evident  granules  (cf. 


SPECIAL   METHODS. 


159 


Fig.  33,  ///),  which  are  held  together  by  the  mucilage  sepa- 
rated with  them  and  finally  thrown  off  with  them. 

This  expulsion  may  be  caused  by  various  precipitates. 
A  very  suitable  one  is  chrome  yellow  (PbCrOJ,  which  is 
precipitated  in  the  membranes  by  placing  the  algae,  held 
together  by  a  thread,  in  a  .25^  solution  of  potassium  chro- 
mate  (K^CrOJ,  then  rinsing  quickly  in  water,  and  finally 
transferring  them  to  a  .25^  solution  of  lead  acetate.  To 
obtain  a  heavy  precipitate,  this  proceeding  may  be  several 


Fig.  ^3. — /,  membrane  and  gelatinous  sheath  of  Zygnenia  sp.  (X  580).  //,  two  Zygnema- 
cells  after  deposit  of  chrome  yellow  (X  245).  ///,  membrane  and  gelatinous  sheath  of 
Zygnetna  after  deposit  of  chrome  yellow  (X  245).  IV,  the  same  of  Pieurot^nium  Tra- 
becular after  staining  with  fuchsin  (X  950).  F,  the  same  of  Staurastrum  bicorne,  after 
staining  with  gentian  violet  (X  950).  z,  cell-membrane;  ^,  gelatinous  sheath.  /  to  /// 
after  Klebs;  /Fand  F  after  Hauptfieisch. 


times  repeated.  But  the  expulsion  takes  place  the  more 
rapidly  the  less  chrome  yellow  is  deposited,  and  may  not  be 
completed  for  several  days  if  the  deposit  be  large. 

Finally,  it  may  be  remarked  that  this  expulsion  is  not 
directly  dependent  on  the  life  of  the  protoplasm,  and  may 
occur  in  dead  individuals,  under  some  circumstances. 

281.  Klebs  has  also  established  the  remarkable  fact  that 
the    gelatinous    sheaths    increase   markedly  in  density  in  a 


[6o  ^^^^ANICAL   MICROTECHNIQUE. 

solution  of   glucose  and  peptone  by  the  deposit  of  a  sui 
stance  whose  composition  is  not  yet  known. 

This  "  thickening  "  of  the  gelatinous  sheaths  occurs,  how- 
ever, only  when  soluble  albuminoids  and  a  sugar  are  simul- 
taneously present  in  the  surrounding  fluid,  and,  like  the 
expulsion  described,  is  independent  of  the  life  of  the  proto- 
plasm. 

282.  According  to  the  investigations  of  Hauptfleisch  (I), 
the  gelatinous  formations  of  the  Desmidiacece  consist,  on  the 
other  hand,  of  single  prisms  or  caps,  each  of  which  covers  a 
pore  in  the  cell-wall.  These  pores  are  occupied  by  threads 
of  protoplasm  which  commonly  terminate  externally  in 
globular  swellings  which  penetrate  to  a  greater  or  less  dis- 
tance into  the  gelatinous  covering,  in  different  species\(cf. 
Fig.  33,  /Fand  V), 

For  the  observation  of  these  structural  relations,  this 
author  recommends  that  at  first  dilute,  and  then  gradually 
more  concentrated  solutions  of  safranin,  fuchsin,  gentian 
violet,  methylene  blue,  or  methyl  violet,  be  allowed  to  run 
from  the  edge  to  the  living  algai  under  a  cover-glass,  and 
that  the  changes  in  the  jelly  during  the  action  of  the  stain 
be  followed.  Then  the  changes  may  be  followed  backward 
by  careful  washing  of  the  specimens. 

The  presence  of  two  different  substances  in  the  gelat- 
inous covering  has  been  disputed  by  Hauptfleisch  for  tl.e 
Desmids. 

5.  Fungus-cellulose. 

283.  The  membranes  of  the  fungi  show  very  varying 
relations.  In  a  number  of  species  they  give  the  normal 
cellulose  reactions,  and  this  is  especially  the  case  in  young- 
stages  (cf.  de  Bary  II,  9).  But  in  most  fungi  they  differ 
from  pure  cellulose  membranes  in  being  insoluble  in  cupram- 
monia  and  in  being  colored  only  yellow  or  brown  by  iodine 
and  sulphuric  acid  or  by  chloroiodide  of  zinc.  They  also 
show  great  powers  of  resistance  to  alkalies  and  acids  in 
general.  But  since,  on  the  other  hand,  they  do  not  show  the 
reactions  for  lignification  or  suberization,  we  are  compelled 


SPECIAL    METHODS.  l6l 

at  present  to  regard  them  as  a  special  modification  of  cellu- 
lose, which  is  commonly  termed  fungus-cellulose. 

It  should  be  remarked  that,  according  to  the  researches  of 
K.  Richter  (I),  the  membranes  of  a  large  number  of  fungi 
giv^e  the  reactions  for  pure  cellulose  after  being  first  treated 
for  a  long  time  with  caustic  potash.  But  in  many  cases  the 
caustic  potash  must  act  for  a  week. 

On  the  other  hand,  W.  Hoffmeister  (I,  254)  has  lately  ob- 
tained from  the  fructification  of  Boletus  edidis,  by  the  use  of 
methods  always  successful  with  the  higher  plants,  no  com- 
pounds giving  the  reactions  of  cellulose.  The  membranes  of 
this  fungus  are,  according  to  his  researches,  characterized  by 
being  completely  soluble  in  concentrated  hydrochloric  acid 
and  caustic  potash. 

284.  The  Membranes  of  the  Bacteria. — There  can  now  be 
no  doubt  that  the  Bacteria  possess  a  solid  membrane.  In 
most  cases  its  presence  may  be  readily  demonstrated  by 
plasmolyzing  the  organisms  (cf.  §§  431  and  463). 

No  reliable  statements  can  be  made  at  present  as  to  the 
chemical  constitution  of  these  membranes.  They  seem, 
moreover,  to  consist  in  part  of  cellulose;  at  least,  W.  Hoff- 
meister (I,  253)  has  isolated  a  substance  reacting  like  cellu- 
lose from  a  species  of  Bacillus  not  exactly  determined. 

6.  Paragalactan-like  Substances  (Hemicelluloses). 

285.  Reiss  and  E.  Schulze  have  shown  that,  especially 
in  the  cell-walls  of  seeds  with  considerable  thickenings  of 
the  walls,  carbohydrates  occur  which  differ  essentially  from 
cellulose  and  are  dissolved  at  germination,  like  the  other 
reserve  materials  of  the  seed.  One  of  these  substances  is 
called  by  Reiss  reserve-cellulose,  another,  by  Schulze,  para- 
galactan.  But  it  is  probable  that  various  related  com- 
pounds exist.  All  these  substances  can  at  present  best  be 
grouped  under  the  name  proposed  by  E.  Schulze,  '*  Paraga- 
lactan-like compounds." 

[Schulze's  later  studies  (II)  afford  ground  for  distinguish- 
ing this  group  of  substances  from  cellulose  as  hemicelluloses. 
He  finds  that  they  become  soluble,  with  the  formation  of 


j62  BOTAMCAI.    MJCA'OT/'.CHXIQUE. 

'glucose,  through  the  action  of  hot  dilute  mineral  acids,  by 
which  true  celluloses  are  not  affected.  They  are  dissolved 
by  dilute  alkalies  and  by  cuprammonia  after  brief  treatment 
with  hot  dilute  hydrochloric  acid,  too  short  to  cause  their 
solution.] 

a.   Reiss'  Reserve-cellulose. 

286.  The  so-called  reserve-cellulose  has  been  prepared  by 
Reiss  (I)  from  the  endosperm  of  Phoenix  daciylifera,  Phyt- 
i'lephas,  and  various  other  seeds  with  strongly  thickened 
cell-walls.  It  differs  from  the  ordinary  cellulose  especially 
in  the  products  resulting  from  hydrolysis  with  sulphuric 
acid.  There  is  first  formed  a  compound  corresponding  to 
■dextrine,  but  Isevo-rotary  {seminin),  and  then  a  dextro-rotary 
sugar  which  reduces  Fehling's  solution  and  is  fermentable 
{seininose)  and  especially  characterized  by  the  fact  that 
It  forms  with  phenylhydrazin  acetate  (C6Hj,NH.NH2)  an 
hydrazon,  which  may  be  obtained  in  crystalline  form,  of  the 
composition  C^Hj^N^Oj,  probably  according  to  the  reaction: 
C.H.,0,  +  QH,N,  =  C„H„0,N,  +  H,0.  Reserve-cellulose 
cannot  be  distinguished  microchemically  from  ordinary  cel- 
lulose and  behaves  quite  like  pure  cellulose  with  iodine  solu- 
tions and  cuprammonia. 

An  exception  is  shown  only  by  the  cell-walls  of  the  endo- 
sperm of  Paris  qiiadrifolia  and  Fceniculum  officinale,  which 
are  insoluble  ifi  cuprammonia,  although  they  give  seminose 
on  hydrolysis   and   must  therefore  be  regarded  as  reserve 
cellulose. 

[Schulze  finds  (II)  that  this  substance  shows  the  characters 
of  other  hemicelluloses  (cf.  §  285)  and  should  be  placed 
.among  them,  with  the  name  mannose.'] 

b.  Paragalactan. 

287.  The  name  paragalactan  has  been  given  by  E.  Schulze 
(cf.  Schulze  I,  and  Schulze,  Steiger,  and  Maxwell.  I)  to  a 
compound  recognized  in  the  thickenings  of  the  walls  of  the 
cells  of  the  cotyledons  of  Liipimis  luteus,  with  true  cellulose, 
and  which  very  probably  occurs  in  other  Legumitiosa:.     It 


SPECIAL    METHODS.  J63 

yields,  on  oxidation  with  nitric  acid,  mucic  acid  ;  on  heating 
with  dilute  sulphuric  acid,  galactose  (CeHj^Og)  and  a  penta- 
glucose.  It  is  also  characterized  by  giving  a  cherry-red 
fluid  on  heating  with  phloroglucin  and  hydrochloric  acid, 
while  no  color  is  produced  in  the  cold.  On  heating,  para- 
galactan  is  transformed  by  i^  hydrochloric  acid  or  i^  sul- 
phuric acid  into  sugar,  while  cellulose  is  attacked  only  by 
pretty  concentrated  solutions. 

It  is  an  important  fact  for  the  microchemical  recognition 
of  paragalactan  that  it  is  insoluble  in  cuprammonia  and 
prevents  the  solution  of  the  cellulose  which  occurs  in  the 
same  membranes,  while  the  latter  is  readily  dissolved  by 
'Cuprammonia  after  the  removal  of  the  paragalactan  by 
boiling  2.5^  hydrochloric  acid.  Paragalactan  does  not  seem 
to  be  colored  by  chloroiodide  of  zinc  ;  at  least,  membranes 
treated  with  this  reagent  showed  only  a  slight  bluing,  while 
the  remains  of  the  membrane  are  deeply  colored  after  the 
solution  of  the  paragalactan. 

[This  substance  also  shows  the  characteristics  of  the 
hemicelluloses  (cf.  §  285).  The  pentaglucose  which  it  yields 
besides  galactose  is  probably  arabinose,  and  it  may  there- 
fore be  called  paragalacto-araban.  It  is  very  possible  that  it 
is  a  mixture  of  two  substances,  galactan  and  araban.] 

c.  Arabanoxylan. 

[287a.  Schulze  finds  (II)  a  hemicellulose  in  wheat  and 
rye  bran  which  yields,  on  hydrolysis,  an  arabinose  and  a 
xylose,  and  may  therefore  receive  the  above  name.] 

7.  Callose,  the  Callus  of  the  Sieve-tubes. 

288.  Until  recently  the  name  callus  was  generally  given 
to  a  pretty  strongly  refractive  substance  which  causes  a 
more  or  less  complete  closing  of  the  sieve-poles  in  old  sieve- 
tubes,  and  finally  covers  the  whole  sieve-plate  with  a  thick 
mass.  Mangin  has  lately  recognized  (I-III)  the  more  gen- 
eral distribution  of  this  substance,  especially  in  the  mem- 
branes of  various  pollen-grains  and  pollen-tubes  and  in 
many  fungi.      It  is,  for  instance,  widely  distributed  in  the 


l64  BOTANICAL   MICROTECHNIQUE. 

mycelium  of  the  Pcronosporacece,  where  it  partly  incrusts  the 
cellulose  wails  and  partly  occurs  in  more  or  less  pure  condi- 
tion in  the  interiors  of  the  hypha^  and  of  the  haustoria  (cf. 
Mangin  III).  [The  same  author  has  also  shown  (IX)  the 
presence  of  this  substance  in  various  cell-walls  of  Phanero- 
gams which  are  incrusted  with  carbonate  of  lime,  especially 
those  of  the  cystoliths  of  the  Urticales  and  of  the  calca- 
reous hairs  and  pericarps  of  several  Borraginacece.  In  the 
achenes  of  Lithospenmim,  Cynoglossurn,  etc.,  where  it  occurs 
without  a  deposit  of  lime,  its  occurrence  seems  to  be  related 
to  the  disappearance  of  the  cell-contents  and  the  gradual 
destruction  of  the  parenchyma.  He  has  also  observed  it  in 
the  walls  of  cells  bordering  tissues  which  have  become 
suberized  in  consequence  of  injuries.] 

This  author  calls  this  substance  callose,  a  term  w^hich 
deserves  preference,  since  the  word  "callus"  is  used,  as  is- 
well  known,  in  quite  another  sense. 

288a.  Callosc  gives  the  following  reactions,  according  to 
Mangin  (II)  :  It  is  insoluble  in  water,  alcohol,  and  cupram- 
monia,  in  the  latter  even  after  previous  treatment  with 
acids.  But  it  is  readily  soluble  in  a  cold  i^  solution  of 
caustic  soda  or  potash,  and  is  also  soluble  in  the  cold  in 
concentrated  sulphuric  acid,  as  well  as  in  concentrated  solu- 
tions of  calcium  chloride  and  stannic  chloride.  Cold  solu- 
tions of  alkaline  carbonates  and  of  ammonia  make  it  swell 
and  give  it  a  gelatinous  consistency,  but  without  dissolv- 
ing it. 

Callose  also  differs  from  cellulose  in  its  behavior  with 
various  coloring  matters.  Mangin  gives  (V)  a  number  of 
azo-colors  which  deeply  stain  cellulose  in  a  neutral  or  feebly 
acid  solution,  but  leave  callose  uncolored  ;  they  are  espe- 
cially orsciUin  BB,  azorubin,  ftaphiol  black,  and  the  crocci?ts. 
On  the  other  hand,  callose  is  distinguished  by  its  strong; 
staining  capacity  with  coralliii  and  aniline  blicc  and  certain 
dyes  belonging  to  the  benzidines  and  tolidines. 

289.  Corallin  or  rosolic  acid  is  best  dissolved  in  a  4^  or 
concentrated  aqueous  solution  of  soda  (Na,CO,).  The  sec- 
tions are  placed  for  a  short  time  in  this  solution  and  then 


SPECIAL   METHODS.  IO5 

examined  in  glycerine,  when,  if  the  staining  has  taken  place 
properly,  the  deep-red  pads  of  callose  stand  out  sharply  in 
the  sieve-tubes.  I  have  found  it  very  useful  to  first  over- 
stain  the  sections  with  corallin  solution  and  then  to  wash 
them  out  with  4^  soda  solution,  which  quickly  decolorizes- 
all  parts  except  the  callose.  This  method  has  given  es- 
pecially good  results  with  the  fungi.  Preparations  stained 
with  corallin  cannot  be  long  preserved. 

290.  Aniline  blue  has  been  recommended  by  Russow  (I)> 
for  staining  sieve-tube  callose.  It  may  be  used  in  a  dilute 
aqueous  solution,  which  is  allowed  to  act  half  an  hour  or 
longer  on  the  sections.  Overstained  sections  may  be  washed 
out  with  glycerine.  They  are  properly  stained  when  only 
the  callose  masses  appear  deeply  colored.  The  •'  Schlauch- 
kopfe"  of  young  sieve-tubes  (§45S)  ^1*^  also  pretty  deeply^ 
colored  by  aniline  blue.  To  distinguish  these  from  the 
callose,  the  preparations  may  be  subsequently  stained  with 
an  aqueous  solution  of  eosin,  in  which  they  are  left  for  a 
few  minutes.  After  a  brief  washing  in  glycerine  the  entire 
contents  of  the  sieve-cells,  including  the  protoplasmic  threads 
which  penetrate  the  sieve-plates,  are-  colored  violet  or  red^ 
while  the  callose  pads  remain  deep  blue.  These  prepara- 
tions, as  well  as  those  with  aniline  blue  ^lone,  can  be  well 
preserved  in  glycerine-gelatine  ;  or  they  may  be  transferred 
to  Canada  balsam  in  the  usual  way. 

Since  it  often  stains  the  protoplasm  pretty  deeply,  aniline 
blue  has  usually  given  me  much  less  instructive  preparations 
than  rosolic  acid  with  fungi. 

[290a.  Mangin  recommends  (IX),  for  staining  the  callose 
of  calcified  membranes,  a  mixture  of  soluble  blue  extra  6B- 
and  vesuvin,  or  of  the  same  blue  and  orseillin  BB.  These 
mixtures  stain  callose  blue  in  a  short  time,  the  protoplasm 
and  lignified  elements  being  brown  or  violet,  according  to 
the  mixture  used.  Where  incrustations  are  not  numerous^ 
as  on  many  leaves,  large  pieces  of  tissue  may  be  freed  from 
air  by  boiling  alcohol,  then  placed  in  cold  nitric  acid  until 
frothing  ceases,  then  in  cold  water,  in  boiling  alcohol,  and 
finally  in   cold  ammonia,  to  remove  xanthoprotein  and  its. 


l66  BOTANICAL   MICROTECHiYIQUE. 

derivatives.  When  the  tissue  is  transparent  enough,  the 
ammonia  may  be  neutraHzed  with  acetic  acid,  and  the  tissue 
placed  in  the  staining  fluid.] 

291.  The  behavior  of  callose  with  iodine  reagents,  which 
has  been  exactly  determined  only  for  the  callose  pads  of 
sieve-tubes,  is  also  characteristic,  and,  according  to  Lecomte 
(I,  268),  best  brings  out  their  intimate  structure.  Chloro- 
iodide  of  zinc  stains  callose  brick-red  or  red-brown  according 
to  the  proportion  of  iodine  it  contains,  calcium  chloride  and 
iodine  solution  (cf.  §  246,  5)  stains  it  rose-red,  or  wine-red 
after  previous  staining  with  aniline  blue,  while  the  sieve- 
plates  are  colored  violet. 

8.  Pectic  Substances. 

292.  Mangin  (IV-VI)  has  lately  shown  microchemically 
that  pectic  substances  (pectin,  pectose,  pectic  acids)  are  very 
widely  distributed  in  the  cell-walls  of  the  most  different 
plants,  and  that  they  form  especially  the  so-called  intercel- 
lular substance  of  unlignified  and  unsuberized  membranes. 

293.  For  the  microchemical  recognition  of  pectic  sub- 
stances Mangin  uses  (IV  and  VI)  chiefly  various  coloring 
matters,  phetiosafrafiin,  methylene  blue,  Bismarck  brown, 
fnc/isin,  Victoria  blue,  violet  de  Paris  (=  methyl  violet  B), 
and  rosolan  (=  mauvcin),  and  others.  These  do  not  color 
pure  cellulose,  but  do  stain  pectic  substances,  as  well  in 
neutral  solution  as  after  slight  acidification  with  acetic  acid. 
But  lignified  and  suberized  membranes  are  also  stained  by 
these  dyes.  However,  there  remains  a  distinction  between 
them  and  pectic  substances  in  that  the  latter  are  quickly 
decolorized  by  alcohol,  glycerine,  and  acids,  while  the  for- 
mer retain  their  color  in  these  fluids.  Mangin  also  gives  a 
number  of  dyes  which  leave  pectic  substances  uncolored  in 
a  neutral  solution,  while  they  deeply  color  lignified  and 
suberized  walls.  Such  colors  are  :  acid  green,  acid  brown, 
nigrosin,  indulin,  the  croceins,  and  the  ponceaux.  Mangin 
obtained  instructive  double  stainings  by  mixing  one  of  these 
dyes  with  one  of  the  previous  group. 

On  the  other  hand,  Mangin  (V)  has  lately  named  a  num- 


SPECIAL   METHODS.  16/ 

ber  of  dyes  which  leave  pectic  substances  uncolored,  but 
stain  cellulose  or  both  cellulose  and  callose.  To  the  former 
belong  orseille  red  A,  naphtol  black,  and  the  croceins ;  while 
Congo-red,  azo-blue,  and  benzopurpurin  stain  cellulose  and 
callose. 

294.  In  order  to  show  that  the  first  described  stainings 
really  depend  upon  the  presence  of  pectic  substances,  Man- 
gin  treated  thin  sections  for  24  hours  with  cuprammonia 
and  then  washed  them  in  water  and  in  2^0  acetic  acid.  On 
this  treatment  the  cellulose  is  removed  from  the  membranes 
and  fills  the  intercellular  spaces  and  the  cell-cavities  as  a 
gelatinous  mass.  In  consequence  of  it  the  membranes  are 
colored  not  at  all  or  but  slightly  yellow  on  the  addition  of 
chloroiodide  of  zinc,  while  a  deep  blue  color  appears  in  the 
interiors  of  the  cells.  The  membranes,  which  now  consist 
of  pure  pectic  acid,  are,  however,  deeply  stained  by  safranin 
or  methylene  blue.  It  is  sufficient  to  add  a  few  drops  of 
a  solution  of  ammonium  oxalate  to  cause  the  solution  of 
the  pectic  acid  membranes. 

295.  In  order  to  show  that  the  middle  lamella  of  the 
so-called  cellulose  membranes  consists  of  pectic  acid  or  an 
insoluble  salt  of  it,  Mangin  (VI)  lets  a  mixture  of  one  part 
hydrochloric  acid  and  4  to  5  parts  alcohol  act  for  24  hours 
on  thin  sections,  then  washes  them  with  water,  and  treats 
them  with  a  weak  (about  10^)  solution  of  ammonia.  After 
this  has  acted  a  short  time,  the  sections  may  be  separated 
into  their  constituent  cells  by  gentle  pressure.  Mangin 
explains  this  by  the  supposition  that  the  pectic  acid  is  set 
free  from  its  originally  insoluble  compounds  by  the  action 
of  the  acid-alcohol,  and  is  then  dissolved  by  the  ammonia 
solution.  In  fact,  a  gelatinous  mass  is  precipitated  from  the 
ammoniacal  solution  on  the  addition  of  acid,  which  has  all 
the  characters  of  pectic  acid.  On  the  other  hand,  sections 
which  were  placed  in  lime-  or  baryta-water  after  the  action 
of  the  acid-alcohol,  showed  no  separation  into  their  cells  on 
subsequent  treatment  with  ammonia,  because  the  pectic 
acid  had  recombined  into  an  insoluble  salt  with  the  alkaline 
earth. 


1 68  BOTANICAL   MICROTECHXIQUE. 

296.  Mangin  (VI)  obtained  a  deep  staining  of  the  middle 
lamella  on  placing  thin  sections  of  adult  plant-organs  in 
i)henosafranin  or  methylene  blue  after  treatment  with  the 
above  mentioned  acid-alcohol  mixture.  The  middle  lamella 
of  pectic  acid  stains  much  more  deeply  than  the  pectic  com- 
pounds mixed  with  cellulose  of  the  thickenings  of  the  cell- 
wall. 

9.  Ash-  and  Silica-skeletons  of  the  Cell-wall. 

297.  The  inorganic  salts  which  incrust  all  vegetable  cell- 
Avalls  are  in  many  cases  present  in  such  quantity  that,  after 
the  destruction  of  all  organic  substances  by  burning,  they 
still  preserve  the  form  of  the  original  membranes. 

Such  ash-skeletons  may  easily  be  obtained  by  burning 
•cross-sections  of  the  stem  of  Citcurbita  Pepo  on  the  cover- 
^lass.  But  they  must  be  examined  in  the  air,  as  they  are  at 
least  partly  soluble  in  water.  These  ash-skeletons  consist 
chiefly  of  potassium  and  calcium  salts.  In  other  cases  silicic 
acid  also  occurs  deposited  in  great  quantity  in  the  mem- 
branes.    For  methods  of  recognizing  this,  see  §§  78-81. 

10.  On  the  Developmental  History  of  the  Cell-wall. 

297a.  In  the  study  of  the  growth  of  cell-walls  it  is  often 
important  to  stain  the  membranes  without  affecting  the 
vitality  of  the  cells.  If  the  objects  thus  treated  are  then 
allowed  to  develop  further  in  pure  water,  it  would  seem  pos- 
sible to  distinguish  the  newly  formed  membranes  or  parts 
of  membranes  from  those  previously  formed,  with  certainty. 

Noll  (I)  proceeded,  with  this  object,  with  Caiilerpa  and 
some  other  marine  algae  by  producing  a  precipitate  of  Ber- 
lin blue  or  TurnbuU's  blue  in  the  membranes  of  the  plants 
under  investigation  without  injuring  their  vitality,  and  then 
allowing  them  to  grow  more,  under  favorable  conditions. 
The  newly-formed  membranes  must  then,  plainly,  be  color- 
less \  and  those  which  have  grown,  perhaps  by  intussuscep- 
tion, must  show  a  lighter  color. 

297b.  To  produce  a  precipitate  of  Berlin  bltie  in  the 
membranes,  Noll  (I,  iii)  placed  the  algae  first,  for  one  or  a 


SPECIAL    METHODS.  1 69 

few  seconds,  in  a  mixture  of  one  part  sea-water  and  two 
parts  fresh  water  to  which  was  added  enough  potassium 
ferrocyanide  to  give  the  solution  the  specific  gravity  of  sea- 
water.  Then  the  algae  were  rapidly  passed  through  a  vessel 
of  pure  sea-water  and  placed  for  one  half  to  two  seconds  in 
a  mixture  of  two  parts  sea-water,  one  part  fresh  water,  and  a 
few  drops  of  ferric  chloride.'^  The  depth  of  the  coloring  is 
increased  markedly  by  repeating  the  proceeding  several 
times. 

For  producing  Turnbiiirs  blue,  which  Noll  thinks  less 
suitable,  he  used  the  corresponding  solutions  of  potassium 
ferricyanide  and  ferrous  lactate. 

297c.  But  it  should  be  remarked  concerning  these  stain- 
ings  that  they  are  gradually  destroyed,  probably  by  the 
excretion  of  alkali.  But  they  can  be  renewed  at  any  time 
by  placing  the  algae  in  a  solution  of  potassium  ferro-  (or 
ferri-)  cyanide  acidified  with  pure  hydrochloric  acid. 

Finally  it  may  be  observed  that,  according  to  Noll's 
researches,  the  vitality  of  the  algae  is  not  destroyed  by 
these  manipulations  and  the  precipitate  is  in  this  way  very 
uniformly  deposited  in  the  'membranes,  provided  they  pre- 
sent no  chemical  differences,  so  that  they  show  the  same 
depth  of  color  in  all  the  layers. 

297d.  Zacharias  (IV,  488)  has  lately  used  Congo  red  in  the 
same  manner  as  Berlin  blue.  He  worked  with  root-hairs  of 
Lepidiiim,  which  he  placed  for  15-30  minutes  in  a  solution 
of  Congo  red  in  water  from  the  public  supply  and  then 
allowed  to  grow  further  in  moist  air.  But,  since  a  decom- 
position of  Congo  red  takes  place  in  light,  the  seedlings 
must  be  cultivated  in  the  dark. 

297e.  Congo  red  was  earlier  used  by  Klebs  (III,  502)  in 
the  investigation  of  the  growth  of  the  membranes  of  various 
algae.  This  author  found  that  Congo  red  has  the  remark- 
able property  of  leaving  membranes  already  formed  color- 
less  01    almost    so,  while  it   gives   a   red   color  to  forming 


*  This  solution  must  be  freshly  prepared  each  time  it  is  used,  as  it  de- 
composes in  a  short  time. 


I/O  BO  TA  NIC  A  L   MICRO  TECIINIQ  UE. 

membranes.  Klebs  used  in  these  studies  a  .01^  solution  of 
Congo  red  or  a  suitable  culture  fluid  to  which  the  same  pro- 
portion of  the  dye  (i  :  10,000)  was  added.  But  it  should  be 
observed  that  the  Congo  red  deposited  in  the  membranes 
strongly  hindered  their  superficial  growth  in  Klebs'  experi- 
ments, while  their  growth  in  thickness  was  so  much  the 
more  increased,  and  the  vitality  of  the  cells  was  in  no  wise 
destroyed. 

II.  The  Finer  Structure  of  Cell-walls. 

297f.  Many  cell-membranes,  especially  those  of  consider- 
able thickness,  are  well  known  to  be  made  up  of  various 
lamellae  or  layers  parallel  to  their  surfaces  {stratification). 
In  many  there  occur  band-like  differentiations  within  the 
same  layer,  which,  according  to  Correns  (III,  324),  always 
have  a  spiral  course  {striation).  Finally,  one  finds  not 
uncommonly  radially  arranged  lamellas  of  varying  optical 
properties  {transverse  iaineilation). 

The  observation  of  these  differentiations  may  in  many 
cases  be  conducted  on  the  unchanged  membranes.  But  in 
general  they  come  out  much  more  plainly  if  the  membranes 
are  treated  with  swelling  media  ;  and,  besides  those  men- 
tioned in  §  10,  chloroiodide  of  zinc  is  in  many  cases  very 
useful. 

297g.  Three  factors  may  enter  into  the  problem  of  the 
cause  of  the  optical  appearances  described,  which  have  been 
thoroughly  discussed  by  Correns  (III):  I.  Sculpturing  of 
the  wall ;  II.  Differentiation  of  the  wall  into  strips  or  layers 
of  unequal  water-content  with  similar  chemical  constitution  ; 
and  III.  Differentiations  of  the  wall  which  possess,  with 
similar  water-content,  unequal  refractive  power,  and  there- 
fore depend  upon  material  differences.  Besides  these,  only 
combinations  of  these  three  factors  are  possible. 

297h.  Sculpturing  of  the  wall  may  produce  especially  stri- 
ation. This  then  falls  into  the  category  of  partial  thicken- 
ings of  the  membrane,  and  deserves  to  be  considered  here 
only  because,  when  very  delicate,  it  cannot  be  distinguished 


SPECIAL    METHODS.  l/I 

from  true  differentiations  of  the  membrane  without  much 
difficulty,  and  often  accompanies  these. 

Striation  due  to  sculpturing  of  the  membrane  is,  plainly, 
only  visible  when  the  membrane  and  the  mounting  fluid 
have  different  refractive  indices.  And  it  becomes  the 
plainer  as  this  difference  is  the  greater,  disappearing  en- 
tirely as  the  refractive  indices  become  equal.  For  example,, 
Canada  balsam  has  almost  the  same  optical  density  as  cell- 
walls  ;  on  the  other  hand,  Correns  used  methyl-alcohol  with 
good  results,  on  account  of  its  low  refractive  index.  This 
is  only  1. 321  and  therefore  less  than  that  of  water  (1.336). 

2971.  Further,  it  is  evident  that  it  is  unimportant  in  case 
of  differentiations  depending  wholly  on  sculpturing  of  the 
wall,  in  opposition  to  those  which  are  to  be  referred  exclu- 
sively to  unequal  water-content,  whether  the  membranes, 
are  placed  in  a  mounting  fluid  of  similar  refractive  index  in 
a  dry  or  swollen  state.  But  the  use  of  this  criterion  en- 
counters difficulties,  as  Correns  has  shown  (III,  260),  if  rifts 
or  canals  in  the  interior  of  the  membrane  are  involved,  as  in 
the  bast-cells  of  Nerhun,  where  the  different  layers  have 
different  systems  of  striation.  In  this  case  it  does  not  seen> 
practicable  to  fill  these  capillary  spaces  with  ethereal  oils  or- 
with  balsam  without  the  removal  of  imbibed  water..  But 
even  in  this  case,  the  behavior  of  the  dried  membranes  on 
being  imbedded  in  Canada  balsam  or  the  like  may  permit 
positive  conclusions  as  to  the  nature  of  the  differentiations; 
in  question,  since  only  a  slow  expulsion  of  the  enclosed  air 
from  capillary  spaces  in  the  interiors  of  membranes  can  take- 
place. 

For  distinguishing  water-bearing  clefts  from  substances 
rich  in  water,  chloroiodide  of  zinc  and  various  dyes  may  be 
used.  Clearly,  the  capillary  spaces  must  always  stand  out  as 
colorless  streaks  on  suitable  sections,  while  the  parts  richer 
in  water  may  show  a  more  or  less  deep  stain. 

297k.  Differentiations  due  to  unequal  zvater-content  must, 
plainly,  disappear  on  drying,  as  a  rule.  The  presence  of 
such  differentiations  may  therefore  be  recognized  by  exam- 
ining the  objects  in  the  same  anhydrous  mounting  fluid  (sucK 


172  BOTANICAL   MICROTECHNIQUE. 

as  Canada  balsam),  a  part  dry  and  a  part  moist.  But  it 
should  be  observed  that  the  complete  removal  of  water  can 
only  be  accomplished  by  drying  at  a  temperature  of  50°  to 
lOO**  C,  according  to  the  nature  of  the  object.  The  deh)-- 
drating  media  used  by  various  authors  for  the  same  purpose, 
especially  absolute  alcohol,  do  not  give  demonstrative  results 
(cf.  Zimmermann  I,  87). 

297I.  It  is  also  to  be  noticed  that,  where  the  water-content 
is  unequal,  changes  in  form  must  occcur  on  drying,  and 
therefore,  as  Correns  (III,  262)  has  specially  observed,  a  cer- 
tain distinction  between  differentiations  due  to  sculpturing 
of  the  wall  and  those  due  to  unequal  water-content  cannot 
always  be  drawn  from  the  comparison  of  dried  and  moist 
membranes.  Concerning  the  possibilities  in  this  respect, 
Correns'  work  (III)  may  be  consulted. 

297m.  The  presence  of  differences  in  water-content  was 
demonstrated  by  Correns  (III,  294)  by  impregnating  the 
membranes  with  a  salt-solution  (NaCl),  which  is  not  recog- 
nizably accumulated.  The  conclusion  is  then  justified  that 
where  the  salt  occurs  in  greater  quantity  this  is  in  conse- 
<juence  of  greater  water-content.  Correns  used  for  this 
purpose  potassium  ferrocyanide  and  silver  nitrate,  and  made 
the  salt  contained  in  the  walls  visible  by  conversion  into 
a  colored  precipitate. 

Concerning  the  method  of  using  the  potassium  ferrocy- 
anidct  it  may  be  observed  that  Correns  placed  the  mem- 
branes, previously  washed  in  water  and  then  dried  b\' 
Avarming  to  50°  to  100°  C,  for  a  few  minutes  in  a  10^ 
solution  of  the  substance  and  then  placed  them,  after  super- 
ficial drying  on  filter-paper,  but  without  washing,  in  a  dilute 
solution  of  ferric  chloride,  in  which  the  formation  of  Berlin 
blue  at  once  takes  place.  In  the  bast-cells  of  Nerium  or 
Vinca,  which  are  especially  suited  to  these  experiments,  the 
striation  becomes  clearly  visible  with  this  treatment,  even 
after  drying  and  mounting  in  cedar-oil  and  Canada  balsam. 

Correns  (III,  295)  was  able  to  show  that  the  potassium 
ferrocyanide  is  merely  absorbed,  but  not  accumulated,  by 
placing  dry  starch-grains  in  a  solution  of  this  salt,  and  testing 


SPECIAL    METHODS.  173 

its  concentration  before  and  after.  It  was  thus-  shown  that 
no  marked  changes  in  concentration  were  produced  by  the 
absorption  of  the  starch.  It  had  previously  been  shown 
by  Sachs  that  potassium  ferrocyanide  rises  with  the  same 
rapidity  as  the  water  in  a  strip  of  filter-paper. 

297n.  For  ''silvering,''  Correns  used  essentially  the  meth- 
ods which  have  been  employed  for  a  long  time  in  animal 
anatomy.  He  placed  the  previously  well-dried  objects  first 
in  a  2-5^  solution  of  silver  nitrate,  and  then,  after  super- 
ficial drying,  but  without  washing  them,  in  a  .75^  solution  of 
salt  (NaCl).  The  silver  chloride  thus  precipitated  in  the 
membranes  is  best  reduced  by  light,  for  which  a  few  hours 
in  direct  sunlight  are  sufficient.  The  objects  are  then  dried 
and  mounted  in  Canada  balsam.  There  may  then  be  ob- 
served in  the  parts  rich  in  water  a  strong  blackening  due  to 
the  reduced  silver,  which  also  forms,  in  part,  small  opaque 
granules.  Here  again  the  bast-cells  of  Neritim  and  Vinca 
are  to  be  recommended,  but  they  should  first  be  washed 
some  time  with  water  for  the  removal  of  silver-reducing 
substances. 

According  to  Correns  (III,  296),  silver  nitrate  is  accumu- 
lated to  a  slight  extent.  A  slight  change  in  the  concentra- 
tion of  the  solution  is  produced  by  starch,  and  the  silver  salt 
remains  somewhat  (about  ^^  behind  the  water  in  filter- 
paper. 

2970.  Differentiations  of  the  membrane  due  to  chemical 
differences  are  especially  recognizable  by  the  fact  that  they 
are  to  be  seen  \\\  the  membranes  whether  dry  or  full  of 
water.  Suitable  objects  for  the  study  of  this  group  of  differ- 
entiations are  furnished  by  the  large  pith-cells  of  Podocarpus 
xlongatus  and  other  species  (cf.  Zimmermann  I,  149). 

The  so-called  transverse  lamellation  of  the  bast-cells  is 
•due  partly  to  chemical  differences  and  partly  to  unequal 
water-content,  according  to  Correns  (II,  298).  The  chemical 
difference  is  shown  by  the  fact  that  the  radial,  more  strongly 
refractive  lamellae  remain  unstained  in  a  pretty  concentrated 
solution  of  methylene  blue,  while  the  rest  of  the  substance  of 
the  wall  is   deeply  stained   by  it.     The   stronger   refractive 


1/4  BOTANICAL   MICROTECHNIQUE. 

power  is  partly  destroyed  by  Schulze's  macerating  mixture^ 
evidently  by  the  solution  of  the  substance  which  causes  the 
stronger  refraction  ;  and  correspondingly,  the  membranes  so 
treated  are  evenly  stained  throughout  by  methylene  blue. 

297p.  Finally,  the  carbonization  or  pulverization  methods 
introduced  by  Wiesner  (V)  into  botanical  microscoi^y  may 
be  here  described.  By  these  the  vegetable  cell-wall  is 
broken  up  into  filamentous  and  then  into  spherical  bodies, 
which  are  called  by  this  author  dermatosomes,  but  whose 
significance  cannot  be  discussed  here. 

Linen  fibres  may  serve  as  suitable  objects  for  the  trial  of 
pulverization  methods.  These  are  laid,  according  to  Wies- 
ner (III,  14),  for  24  hours  in  a  l^  solution  of  hydrochloric 
acid,  then  freed  from  adhering  fluid  and  warmed  to  50°  or 
60°  C,  until  the  substance  is  completely  dry,  which  can  be 
accomplished  in  30  to  50  minutes  if  small  quantities  of  fibre 
are  used.  The  fibre  may  then  be  broken  into  an  extremely 
fine  powder  by  gentle  pressure. 

With  other  objects  a  longer  stay  in  hydrochloric  acid,  or 
the  use  of  higher  temperatures  for  drying,  is  necessary. 
Wiesner  accomplished  the  pulverization  of  endosperm-cells 
of  Phytelephas  only  after  the  action  of  hydrochloric  acid  for 
months. 

Pfeffer  (IX)  has  lately  shown  that  the  carbonization 
methods  lead  to  the  same  results  with  artificially  prepared 
collodion  membranes. 


B.  The  Protoplasm  and  Cell-sap. 

298.  Conclusions  concerning  the  morphological  characters 
of  the  protoplasm  have  been  sought  for  not  only  by  direct 
observation  of  living  material,  but  also  by  means  of  micro- 
chemical  reactions  and  staining  methods.  Although  it  would 
seem  probable  a  priori  that  microchemistry  would  prove 
an  aid  of  the  first  importance,  it  has  not  yet  justified  these 
expectations ;  which  is  due  largely  to  the  fact  that  macro- 
chemical  studies  of  the  structures  in  question  have  been 
carried  through  with  any  exactness  in  very  few  cases,  since 


SPECIAL   METHODS,  1/5 

they  have  to  overcome  great  difficulties  in  the  small  size 
and  ready  decomposition  of  the  bodies  concerned.  There- 
fore it  is  not  yet  certain  whether  the  various  organs  of  the 
plasma-mass,  like  the  nucleoli  or  the  leucoplasts,  consist 
always  of  the  same  or  only  of  related  chemical  compounds  ; 
although  their  behavior  with  certain  staining  agents  makes 
the  former  supposition  seem  probable  for  many  cases.  For 
Ave  possess  certain  staining  methods  which  act  with  such 
precision  as  to  render  them  worthy  of  places  with  the  best 
microchemical  reactions,  and  to  assure  the  first  place  in 
the  investigation  of  protoplasmic  structures  to  staining 
methods.  But  it  cannot  be  doubted  that  in  the  immediate 
future  microchemical  reactions  may  be  of  the  greatest 
importance  in  the  study  of  the  plasma-body. 

In  the  following  pages  we  will  first  discuss  the  methods  of 
recognizing  the  various  inclusions  and  differentiations  of 
the  protoplasm,  and  then  describe  some  methods  which 
have  been  used  in  the  study  of  various  general  character- 
istics of  the  plasma-mass  and  the  cell-sap,  and  of  certain 
physiological  processes. 

I.  The  Nucleus  and  its  Constituents. 

299.  The  advances  which  our  knowledge  of  the  morpho- 
logical characters  of  the  nucleus  have  made  in  recent  dec- 
ades are  almost  exclusively  due  to  staining  methods,  which 
makes  explicable  the  fact  that  the  most  various  natural  and 
artificial  dye-stuffs  have  been  tested  as  to  their  applicability 
in  this  respect,  and  that  innumerable  staining  methods  have 
been  most  warmly  recommended  by  their  discoverers. 

It  cannot  be  necessary  for  me  to  enumerate  all  these 
methods  here,  but  rather  to  limit  myself  to  the  best  of  them, 
which  are  capable  of  general  application  and  do  not  fail  in 
difficult  cases.  I  begin  with  the  enumeration  of  the  most 
important  fixing  and  staining  methods,  and  add  some  gen- 
eral remarks  as  to  the  staining  of  the  nucleus  in  various 
cells  and  on  the  recognition  of  its  various  constituents. 


1/6  BOTANICAL    MICROTECHNIQUE. 

I.   The  various  Methods,  in  general. 

a.  Fixing  Methods. 
a.  Alcohol,  CHiOH. 

300.  Exposure  for  24  hours  is  usually  sufficient  for  fixing- 
nuclei,  but  a  longer  action  does  no  harm. 

If  one  desires  to  prepare  sections  free-hand  from  alcoholic 
material,  it  is  often  useful  to  place  the  material  for  24  hours 
previously  in  a  mixture  of  equal  volumes  of  alcohol  and 
glycerine,  or  of  alcohol,  glycerine,  and  water,  which  makes 
them  much  better  adapted  to  cutting. 

Concerning  the  addition  of  sulphurous  acid  to  such  prep- 
arations as  blacken  in  pure  alcohol,  see  §  34. 

ft.   Iodine. 

301.  Iodine  has  been  chiefly  used  for  fixing  in  an  aqueous 
solution  of  iodine  and  potassium  iodide.  Berthold  (II,  704,. 
note)  recommends  for  marine  alg^e  a  concentrated  solution 
of  iodine  in  sea-water,  which  may  be  prepared  by  the  addi- 
tion of  a  few  drops  of  alcoholic  iodine  solution  to  pure  sea- 
water.  According  to  Berthold,  it  is  sufficient  to  move  the 
algae  about  in  this  fluid  from  half  a  minute  to  a  minute. 
They  are  then  transferred  directly  to  50^  alcohol  and,  if 
this  fluid  be  changed  a  few  times,  can  be  placed  in  the 
staining  fluid  in  a  few  minutes. 

Overton  (I,  530)  has  used  the  vapor  of  iodine,  which  may 
easily  be  obtained  by  warming  iodine-crystals  in  a  narrow 
test-tube,  for  fixing,  with  the  advantage  that  they  may  be 
entirely  expelled  by  gentle  warming  (to  30°  or  40°  C.)  and 
require  no  washing  out  of  the  fixing  medium.  Their  use  is 
especially  to  be  recommended  for  small  objects  (cf.  §  40). 

;'.   Bromine  and  Chlorine. 

302.  The  vapor  of  bromine  is  recommended  by  Stras- 
burger  (I,  399)  for  fixing  Fiicus.  [Zimmermann  (VIII) 
recommends  chlorine  gas  for  fixing  such  algae  as  Cladophorct 
and  Zygnema  without  contraction  of  the  protoplasm.] 


SPECIAL  METHODS,  1/7 

5.   Picric  Acid,  C6H2.{N02)3.0H. 

303.  Picric  acid  is  used  mostly  in  a  concentrated  aqueous 
or  alcoholic  solution.  Its  action  for  24  hours  is  generally 
sufficient.  Before  staining  it  must  be  carefully  washed  out, 
for  which  purpose  running  water  is  especially  useful  (cf. 
§  35).  But  in  many  cases  it  is  better  to  wash  with  alcohol, 
in  which  picric  acid  is  more  readily  soluble  than  in  water. 

e.    Picro-sulphuric  Acid. 

304.  Picro-sulphuric  acid  may  be  prepared,  according  ta 
the  recipe  proposed  by  Mayer,  by  mixing  100  volumes  of 
water  and  two  of  concentrated  sulphuric  acid,  then  shaking 
up  with  it  as  much  picric  acid  as  will  dissolve,  and  finally 
diluting  the  whole  with  three  times  its  volume  of  water. 
Picro-sulphuric  acid  has  the  advantage  over  pure  picric  acid 
that  it  is  more  easily  washed  out.  It  has  been  much  used 
with  the  lower  organisms. 

C.   Chromic  Acid,   H2Cr04. 

305.  Chromic  acid  has  been  used  with  the  best  results  for 
fixing  algae,  especially  in  a  i^  aqueous  solution.  Its  actioa 
for  a  few  hours  is  always  sufficient  for  these  plants  ;  but 
with  larger  tissues  it  is  better  to  allow  the  medium  to  act 
for  24  hours.  Before  staining,  the  chromic  acid  must  always 
be  well  washed  out,  for  which  running  water  is  best  (§  35). 
Overton  recommends  (I,  10)  for  this  purpose  a  weak  aqueous 
solution  of  sulphurous  acid.  This  makes  objects  fixed  in 
chromic  acid  fit  for  staining  with  haematoxylin  and  carmine 
in  a  few  minutes. 

Chromic  acid  has  the  disadvantage  of  often  causing,  espe- 
cially in  tissues  rich  in  tannin,  the  formation  of  precipitates 
which  hinder  observation. 

306.  Finally,  it  may  be  observed  that,  according  to  Vir- 
chow  (I),  objects  fixed  with  chromic  acid  should  be  brought 
in  contact  with  alcohol  only  in  the  dark,  before  the  com- 
plete removal  of  the  acid,  since  their  power  of  staining  is 
lessened  by  the  formation  of  a  precipitate  in  the  light.      In 


.178  BOJANICAL    MICROTECHNIQUE. 

nhe  alcohol  used  for  washing,  a  precipitate  is  also  formed  in 
'the  light;  but  when  this  is  removed  by  filtering  the  fluid 
can  again  be  used  for  washing. 

1],  Chrom-formic  Acid. 

307.  Rabl  recommends  (I,  215)  a  mixture  of  200  grams 
of  Vfo  chromic  acid  with  four  or  five  drops  of  concentrated 
formic  acid  for  fixing  nuclear  figures.  It  must  be  freshly 
prepared  before  use,  each  time,  and  should  act  for  12-24 
hours.  It  must  be  well  washed  out  with  water  before 
staining. 

0.  Osmic  Acid,  OsO*. 

308.  To  fix  sections  or  small  objects,  the  vapor  of  osmic 
acid  may  best  be  used  by  placing  the  objects  in  a  drop  of 
water  on  a  cover-glass  or  slide  and  bringing  the  drop  over 
the  mouth  of  a  bottle  containing  a  \io  or  2^  solution  of 
osmic  acid.  Killing  and  fixing  take  place  almost  instantly. 
For  fixing  larger  pieces  of  tissue  i^o  osmic  acid  may  be  used 
and  may  act  for  several  hours  without  harm. 

Osmic  acid,  which  is  indisputably  one  of  our  best  fixing 
tnedia,  has  the  disadvantage  of  producing  brown  or  black 
precipitates  with  very  various  substances.  But  in  most 
cases  these  precipitates  may  be  removed  subsequently  with- 
out injury  to  the  protoplasmic  structure,  and  for  this  use 
Jiydrogen  peroxide  is  best  adapted.  Overton  (I,  11)  recom- 
mends for  this  purpose  a  mixture  of  one  part  of  commercial 
peroxide  with  10-25  parts  of  70-80^  alcohol.  I  have  also 
observed  that,  even  after  the  use  of  the  concentrated  com- 
mercial solution  of  peroxide,  which  decolorizes  at  once, 
<;specially  on  gentle  warming,  the  karyokinetic  figures  re- 
main quite  sharp  and  unchanged  in  microtome  sections. 

/.   Chrom-osm  ic-acetic  Acid. 

309.  Mixtures  of  chromic  acid,  osmic  acid,  and  acetic 
acid  have  been  used  with  the  best  results  in  the  study  of 
the  karyokinetic  figures,  especially  by  Flemming.  This 
author  has  used  solutions  of  very  different  strengths  ;  but 


SPECIAL   METHODS.  1 79 

the  following,  which  have  both  proved  very  good  with 
plant-cells,  may  be  mentioned  here.  The  first,  dilute  mixt- 
ure contains  .25^  of  chromic  acid,  .1^  of  osmic  acid,  and  .\% 
of  acetic  acid.  The  more  eoncentrated  mixture  is  prepared 
from  15  volumes  of  ifo  chromic  acid,  4  volumes  of  2^  osmic 
acid,  and  one  volume  (or  less)  of  glacial  acetic  acid. 

Both  mixtures  must  be  carefully  u^ashed  out  with  water. 
Blackening  due  to  the  osmic  acid  may  be  removed  with 
hydrogen  peroxide  (cf.  §  308). 

For  preserving  objects  fixed  with  the  above  mixture, 
Flemming  (III,  687,  note)  recommends  a  mixture  of  water, 
alcohol,  and  glycerine,  in  about  equal  parts.  This  affects 
their  staining  capacity  less  than  pure  alcohol. 

K.  Corrosive    Sublimate,    HgClo. 

310.  Corrosive  sublimate  is  usually  best  used  in  concen- 
trated alcoholic  solution,  though  the  concentrated  aqueous 
solution  often  does  well.  Action  for  a  few  hours  is  always 
sufficient  for  complete  fixing,  but  it  may  be  left  on  the 
objects  without  harm  for  24  hours.  Alchohol  to  which 
enough  iodine  has  been  added  to  give  a  dark  brown  solution 
may  be  used  for  washing  out  the  sublimate.  If  it  is  not 
wholly  washed  out,  needle-shaped  or  sphaerite-like  crystals 
of  sublimate  may  be  seen  in  the  preparation  and  may  easily 
-deceive  beginners.  But  where  the  sublimate  has  not  been 
wholly  removed  before  imbedding  in  paraffine,  it  may  subse- 
quently be  washed  out,  even  from  microtome  sections,  with 
the  iodine-alcohol. 

If  it  is  desirable  to  avoid  alcohol  in  washing  out  sublimate, 
the  mixture,  proposed  by  Haug  (I,  13),  of  two  parts  tinc- 
ture of  iodine,  one  part  potassium  iodide,  50  parts  glycerine, 
and  50  parts  water  may  be  used.  It  should  be  renewed 
until  no  further  decolorization  occurs. 

I  will  remark  that  objects  fixed  with  sublimate  must  not 
be  touched  with  iron  forceps  or  the  like  before  it  is  wholly 
washed  out,  since  globules  of  mercury  are  thus  easily  pro- 
duced within  the  objects.  In  this  case  forceps  with  platinum 
or  horn  points  may  be  used. 


l8o  BOTANICAL    MICROTECHNIQUE. 

/I.    Platinum  chloride,    PiCU. 

311.  A  \i)  aqueous  solution  of  platinic  chloride  has  been 
recommended  by  Rabl  (I,  216)  for  fixing  nuclear  figures. 
Its  use  brings  out  especially  the  longitudinal  splitting  of  the 
segments  of  the  nuclear  thread  and  the  chromatin  spheres. 
It  should  act,  in  general,  for  24  hours. 

//.  Chromic-acid-Platinum-chloride. 

312.  Merkel  used  the  following  combination  :  100  volumes 
of  i^  chromic  acid,  100  volumes  of  i^  platinic  chloride,  and 
600  volumes  of  water.  This  medium  renders  good  service 
also  with  vegetable  objects.     It  should  act  for  24  hours. 

V.   Platinum-chloride-Osmic-acetic  acid. 

313.  F.  Hermann  (I,  59)  has  recommended  the  following- 
mixture:  15  volumes  of  a  i^  platinic  chloride  solution,  one 
volume  of  glacial  acetic  acid,  and  two  or  four  volumes  of 
25^  osmic  acid.  Hermann  allows  this  mixture  to  act  from 
one  to  two  days.  To  show  the  achromatic  nuclear  figure, 
he  washes  the  fluid  out  in  running  water,  hardens  in  alcohols 
of  increasing  strength,  and  then  lays  the  objects  in  crude 
pyroligneous  acid  for  12  to  18  hours  (cf.  Hermann,  II,  571). 

b.  Staining  Methods.  , 

a.   Haemato.xylin. 

314.  Besides  carmine,  hrematoxylin  has  been  most  used 
for  staining  nuclei,  and  we  have  a  great  number  of  recipes 
for  the  preparation  of  specially  active  haematoxylin  solu- 
tions. Among  these  the  so-called  Deiafic/d's  hcematoxylin 
solution,  also  often  erroneously  called  Grejiachers  haema- 
toxylin, seems  to  deserve  preference  in  most  cases.  It  is 
prepared  as  follows  :  4  grams  of  haematoxylin  are  dissolved 
in  25  ccm.  of  alcohol,  then  400  com.  of  a  concentrated 
aqueous  solution  of  ammonia  alum  are  added,  and  the  mix- 
ture is  allowed  to  stand  in  the  light  for  3  or  4  days  and  is 
filtered  ;  100  ccm.  of  glycerine  and  100  ccm.  of  methyl  alco- 
hol are  added,  the  whole  is  allowed  to   stand   a^ain    for  a 


SPECIAL   METHODS.  1 8 1 

few  days  and  is  filtered  again.  Since  the  method  of  pre- 
paring it  is  somewhat  complicated,  many  will  prefer  to> 
obtain  it  ready  prepared  from  a  chemist  (e.g.,  from  Dr.  G. 
Griibler,  Leipzig). 

315.  The  so-called  BoJuners  hcematoxylin  is  also  very  useful. 
It  is  prepared  from  a  concentrated  alcoholic  solution  of 
haematoxylin  which  contains  .35  gram  of  haematoxylin  to  lo- 
grams  of  alcohol  and  will  keep  indefinitely.  A  few  drops  of 
this  are  added  to  a  solution  of  .10  gram  of  alum  in  30  ccm. 
of  water.  This  mixture  is  allowed  to  stand  for  a  few  days» 
and  is  filtered  befo;-e  use. 

P.  Mayer  (HI)  obtained  an  haematoxylin  solution  that  may^ 
be  used  at  once  by  dissolving  i  gram  of  hcematein  or  JicEinatein^ 
ammonia  in  50  ccm.  of  90^  alcohol  by  warming,  and  then 
adding  the  whole  to  a  solution  of  50  grams  of  alum  in  a  litre 
of  water.  This  solution  may  be  diluted  with  distilled  water 
for  staining,  as  desired. 

Concerning  the  other  solutions  of  haematoxylin  which  ma)^ 
be  valuable  in  special  cases,  and  may  in  part  be  obtained 
ready  for  use  from  various  chemists,  reference  may  be  had 
to  the  compilation  of  Gierke  (I,  32-35). 

316.  If  it  is  desired  to  stain  sections  with  haematoxylin,. 
they  are  best  placed  in  a  very  dilute  solution  and  left  in  it 
for  a  considerable  time  (i  to  24  hours).  With  alcoholic 
material  it  is  advisable  to  place  it  in  water  for  a  short  time- 
before  staining  it,  as  otherwise  precipitates  are  readily 
formed. 

Beautifully  differentiated  stainings-  may  usually  be  ob- 
tained by  staining  sections  too  deeply  (*'  overstaining")  and 
then  washing  them  out  with  a  suitable  fluid.  With  an 
haematoxylin  stain,  a  solution  of  alum  (about  2^)  is  com- 
monly best ;  but  it  must  be  thoroughly  washed  out  before 
the  transfer  to  alcohol  or  to  Canada  balsam,  as  otherwise 
alum  crystals  will  be  formed  in  the  preparation. 

Acid  alcoJiol  has  also  been  recommended  for  washing  out 
haematoxylin;  but  the  acid  must  be  completely  removed 
with  pure  alcohol  before  the  final  mounting. 

Very  good   nuclear  staining  may  often   be   obtained    by 


]82  BOTANICAL   MICROTECHNIQUE. 

placing  objects  stained  with  ha^matoxylin  for  a  short  time 
in  a  i^  sohition  of  potassiiivi  bichromate  or  a  concentrated 
aqueous  solution  oi  picric  acid.  Both  fluids  must,  of  course, 
be  carefully  washed  out  before  the  transfer  to  Canada  bal- 
sam or  glycerine-gelatine. 

317.  In  dealing  with  objects  to  be  sectioned  with  the 
microtome,  very  pure  nuclear  stains  may  usually  be  obtained 
by  staining  the  objects  in  toto  (**  staining  in  mass")  before 
imbedding  in  paraffine.  Since  haematoxylin  cannot  pene- 
trate the  cuticle  and  therefore  only  penetrates  from  cut 
surfaces,  very  different  depths  of  staining  are  obtained,  and, 
at  some  distance  from  the  original  surface,  only  a  nuclear 
stain.  Large  objects  must  sometimes  be  left  in  the  staining 
fluid,  which  in  this  case  should  be  used  pretty  dilute,  for 
some  time  (often  several  days),  in  order  to  be  sufficiently 
stained.  Very  good  mass-staining  may  be  obtained  by  first 
staining  large  pieces  of  tissue  deeply  with  haematoxylin  and 
then  placing  them  for  a  considerable  time  in  a  i^  solution  of 
potassium  bichromate. 

(i.  Carmine. 

318.  Only  a  few  of  the  numberless  different  carmine 
solutions  can  be  described  in  detail  here.  These,  as  well  as 
various  other  staining  media  containing  carmine,  can  be 
obtained  ready  for  use  from  chemists. 

1.  Grenadier  s  horax-carmine  can  be  prepared  by  dissolv- 
ing 4  grams  of  borax  and  2  to  3  grams  of  carmine  in  93  ccm. 
of  water,  then  adding  100  ccm.  of  70^  alcohol,  shaking  and 
filtering.  This  solution  is  used  for  staining  in  mass  as  well 
-iis  for  sections.  For  washing,  acid  alcohol  and  a  solution  of 
borax  or  oxalic  acid  in  spirit  are  recommended. 

2.  Beales  Carmine. — .6  gram  of  carmine  is  shaken  up  with 
3.75  grams  of  liquor  ammonii  canst,  [aqua  ammoniae,  U.  S.  P.], 
then  boiled  for  a  few  minutes  ;  after  an  hour,  60  grams  of 
glycerine,  60  grams  of  water,  and  15  grams  of  alcohol  are 
added,  and  the  whole  is  finally  filtered. 

3.  Ammonitim  carminate  is  best  prepared  by  dissolving  in 
^vater  to  which  a  little  (about  .5^)  ammonium  carbonate  has 


SPECIAL    METHODS.  1 85 

been  added,  the  commercial  dry  ammonium  carminate  (the 
so-called  Hoyer's  ammonium  carminate).  Alcohol  or  acid 
alcohol  is  best  used  for  washing. 

4.  Sodium  carminate  can  be  obtained  in  solid  form,  and 
may  be  dissolved  in  an  aqueous  .5^  solution  of  ammonium 
carbonate. 

5.  P.  Mayer  s  carmine  solution  is  prepared  by  rubbing  up 
4  grams  of  carmine  in  15  ccm.  of  water,  then  adding  30  drops 
of  hydrochloric  acid  while  warming,  and  finally  adding  95 
ccm.  of  85,^  alcohol,  boiling,  neutralizing  with  ammonia,  and 
filtering  when  cold.  Is  used  both  for  staining  sections  and 
for  staining  in  mass. 

6.  Carminic  Acetate. — Ammonium  carminate  is  decom- 
posed with  acetic  acid  added  drop  by  drop  in  the  least 
possible  excess  until  the  cherry-red  fluid  has  become  brick- 
red,  when  it  is  filtered.  For  washing,  a  mixture  of  one  part 
hydrochloric  acid  in  200  parts  glycerine  or  one  of  one  part 
formic  acid  in  lOO  parts  glycerine  is  recommended. 

7.  Picrocannine  is  the  term  applied  to  variously  prepared 
mixtures  of  picric  acid  and  carmine.  Only  the  simplest 
recipe  (Hoyer's)  need  be  given  here.  According  to  this,, 
pulverized  carmine  is  dissolved  in  a  concentrated  solution 
of  neutral  ammonium  picrate. 

P.  Mayer  (HI)  now  uses  pure  carminic  acid  for  the  prep- 
aration of  carmine  solutions,  of  which  he  especially  recom- 
mends the  following: 

8.  Carmalnni  is  prepared  by  dissolving  i  gram  of  carminic 
acid  and  10  grams  of  alum  in  200  ccm.  of  distilled  water, 
with  heat.  The  solution  may  be  decanted  off  or  filtered. 
To  protect  it  against  decomposition,  this  author  finally  adds 
a  few  crystals  of  thymol,  or  .1^  of  salicylic  acid  or  .5^^  of 
sodium  salicylate.  On  washing  with  water  the  protoplasm 
remains  somewhat  colored.  To  obtain  a  purely  nuclear 
stain,  the  washing  must  be  carefully  done  with  a  solution  of 
alum  or  a  weak  acid. 

9.  Paracarmine  is  prepared  according  to  the  following 
recipe:  i  gram  of  carminic  acid,  \  gram  of  aluminium  chlo- 
ride, and  4  grams  of  calcium  chloride  are  dissolved  in    100 


BOTANICAL   MIL 

•ccm.  of  70^  alcohol  with  or  without  heat,  the  whole 
-allowed  to  stand  and  then  filtered.  Washing  with  aci 
alcohol  is  usually  unnecessary,  but  a  weak  solution  of  alu- 
minium chloride  in  alcohol,  or  alcohol  containing  2^^  of 
dacial  acetic  acid,  is  sufficient  for  all  cases. 

According  to  P.  Mayer's  statements,  only  Grenacher's 
borax-carmine,  of  the  numberless  solutions  heretofore  rec- 
ommended, presents  any  advantages  over  carmalum  and 
paracarmine. 

A.  Meyer  (V)  recommends  especially  for  staining  the 
jiuclei  of  pollen-grains: 

10.  Chloral  Carmine. — This  is  prepared  by  heating  for  30 
minutes  on  the  water-bath  .5  gram  of  carmine,  20  ccm.  of 
-alcohol,  and  30  drops  of  officinal  hydrochloric  acid,^  and 
then  adding  25  grams  of  chloral  hydrate.  After  cooling,  the 
solution  is  filtered.  It  stains  the  nuclei  of  pollen-grains 
<ieep  red  in  ten  minutes.  In  many  cases  I  have  obtained 
good  nuclear  stains  with  this  medium  by  washing  the  sec- 
tions in  boiling  water  on  removing  them  from  it.  Such 
preparations  may  be  mounted  in  glycerine  or  may  be  trans- 
ferred in  the  usual  way  to  Canada  balsam. 

[11.  Czokors  Alum  cochineal  has  proved  so  good  as  a 
nuclear  stain  for  many  vegetable  tissues  that  it  should  have 
a  place  here.  It  is  prepared  as  follows :  7  grams  of  crude 
pulverized  cochineal  (the  dried  insects)  is  boiled  in  a  solu- 
tion of  7  grams  of  burnt  alum  in  700  ccm.  of  water  until  the 
whole  is  reduced  to  400  ccm.  The  solution  is  then  carefully 
iiltered  and  a  few  crystals  of  carbolic  acid  (phenol)  are 
added  as  a  preservative.] 

319.  All  these  solutions  of  carmine  are  adapted  as  well 
for  staining  in  mass  as  for  staining  sections.  But  they  gen- 
erally penetrate  pretty  slowly  and  require  to  act  for  a  rather 
long  time.  They  are  inferior  in  the  sharpness  of  their  stain- 
ing to  many  other  dyes,  in  most  cases ;  but  usually  give  a 
very  purely  nuclear  stain.  The  cell-wall  is  especially  seldom 
stained  by  carmine. 

[*  This  has  a  specific  gravity  of  1.13  or  17°  B.] 


SPECIAL   METHODS.  185 

y.  Safranin. 

320.  The  aniline-water-safranin  recommended  by  Zwaar- 
demaker  (I)  is  best  used  for  staining  with  this  dye.  It  may 
be  prepared  by  mixing  equal  volumes  of  a  concentrated 
alcoholic  solution  of  safranin  and  aniline-water  ^ ;  and 
should  be  allowed  to  act  for  an  hour  or  longer.  Prepara- 
tions may  be  washed  with  alcohol  or  acid  alcohol,  i.e., 
alcohol  containing  about  .5^  of  hydrochloric  acid. 

<5,     Gentian   Violet    (Gram's    Method). 

321.  The  method  originally  proposed  by  Gram  for  staining 
isolated  bacteria  (cf.  §  470)  is  in  many  cases  well  adapted  for 
staining  nuclei,  especially  in  material  which  has  been  fixed 
with  one  of  the  acid  mixtures  recommended  in  §§  309,  312, 
and  313.  It  is  well  to  use  here  as  a  staining  fluid,  a  mixture 
of  3  grams  of  aniline,  i  gram  of  gentian  violet,  15  grams  of 
alcohol,  and  100  grams  of  water.  The  sections  remain  in 
this  one  or  a  few  minutes,  the  stain  is  washed  off  with, 
alcohol,  and  a  solution  of  i  part  of  iodine  and  2  parts  of 
potassium  iodide  in  300  parts  of  water  is  at  once  added. 
This  gives  the  sections  a  dark  color  and  is  then  washed  off 
with  alcohol ;  clove-oil  is  added,  which  extracts  more  color- 
ing matter  from  the  sections,  and  usually  first  brings  out  the 
characteristic  differential  staining ;  finally  the  sections  are 
mounted  in  Canada  balsam. 

It  may  be  especially  remarked  here  that  in  this  process 
the  action  of  .the  clove-oil  is  in  no  way  merely  a  clearing 
one,  as  has  often  been  said.  If  it  be  replaced  by  xylol,  even 
after  thorough  washing  with  alcohol,  not  nearly  so  good 
nuclear  stains  will  be  obtained,  in  many  cases,  as  where  clove- 
oil  is  used. 

Finally,  a  very  good  double  stain  may  be  obtained  by 
dissolving  eosin  in  the  clove-oil  used  for  washing.  The  walls 
and  the  achromatic  nuclear  figure  appear  pure  red,  and  the 
•chromatic  figure  violet. 

*  This  is  prepared  by  shaking  water  with  an  excess  of  aniline,  and  contains 
about  3.5^  of  aniline. 


1 86  BOTANICAL   M ICROTECHXIQUE. 

6.   Safranin    and    Gentian    Violet. 

322.  According  to  the  methods  recommended  by  Her- 
mann  (I,  60),  the  sections  are  first  placed  for  24-48  hours  in 
a  solution  of  safranin  which  contains  i  gram  of  the  dye  to- 
10  ccm.  of  alcohol  and  90  ccm.  of  aniline-water.  They  are 
then  treated  successively  with  water,  acid  alcohol,  and  alco- 
hol, but  so  that  they  remain  still  too  deeply  stained  for 
direct  observation.  From  the  alcohol,  the  sections  arc  then 
transferred  for  3  to  5  minutes  to  a  solution  of  gentian  violet 
containing  i  gram  of  that  dye  to  10  ccm.  of  alcohol  and  9a 
ccm.  of  aniline-water.  The  sections  are  then  quickly  rinsed 
off  in  alcohol  and  placed  in  a  solution  of  iodine  and  potas- 
sium iodide  prepared  as  for  Gram's  method  (§  321),  in  which 
they  remain  for  one  to  three  hours  until  they  are  quite 
black.  Then  they  are  differentiated  in  alcohol,  cleared  in 
xylol,  and  mounted  in  Canada  balsam. 

In  successful  preparations  the  nucleoli  of  resting  nuclei 
are  vivid  red,  the  nuclear  fra'mework  blue-violet.  Of  the 
karyokinetic  figures  the  spirem  and  dispirem  are  blue,  while 
the  intermediate  stages  are  red.  The  achromatic  figure  is- 
lightly  stained  yellow-brown  by  the  iodine. 

C.  Safranin-Gentian- violet-Orange. 

323.  According  to  Flemming  (III,  685,  note),  objects 
fixed  with  chrom-osmic-acetic  acid  (cf.  §  309)  or  with  Her- 
mann's platinum-chloride-osmic-acetic  acid  (cf.  §  313)  may 
best  be  placed  for  2  or  3  days  in  a  concentrated  alcoholic  so- 
lution of  safranin  which  is  diluted  with  about  an  equal  volume 
of  water  and  a  little  aniline-water.  They  are  then  washed  in 
water  and  then  extracted  with  alcohol  containing  at  most  .1;^ 
of  hydrochloric  acid,  or  with  pure  alcohol.  After  a  brief 
washing  in  water,  the  objects  are  placed  for  i  to  3  hours  in  a 
concentrated  aqueous  solution  of  gentian  violet,  and  then, 
after  another  short  washing  in  water,  in  a  concentrated  aque- 
ous solution  of  orange,"*^  in  which  its  dark  color  is  gradually 
dissolved  out.     After  a  few  minutes,  while  blue  clouds  of 

♦  This  may  be  obtained  from  Dr.  G.  Griibler  under  the  name  "  Orange  G." 


SPECIAL   METHODS.  1 8/ 

color  are  still  rising,  the  objects  are  placed  in  neutral  absolute 
alcohol,  and,  after  the  repeated  renewal  of  this,  in  clove-oil 
or  bergamot-oil,  which  still  extracts  light  clouds  of  color, 
and  finally  mounted  in  balsam.  The  difificulty  of  this 
method  consists  in  determining  the  right  minute  when  the 
objects  in  alcohol  and  in  oil  are  neither  too  much  nor  too 
little  washed  out. 

In  successful  preparations  the  chromatin  will  be  purple- 
red,  the  threads  of  the  achromatic  spindle  gray-brown,  gray, 
or  violet,  the  centrosomes  (cf.  §  348a)  the  same  or  light 
reddish. 

324.  I  have  tested  the  last  two  methods  upon  the  root- 
tips  of  Vicia  Faba  and  on  various  other  vegetable  objects 
and  can  most  heartily  recommend  them  for  staining  vege- 
table nuclei.  I  obtained  the  most  instructive  preparations 
by  leaving  the  sections  (I  worked  with  microtome  sections) 
from  j-  to  2  hours  in  Hermann's  aniline-water-safranin.  I 
then  washed  them  successively  in  acid  alcohol  and  in  alcohol,, 
added  aniline-water-gentian-violet,  which  I  left  on  the  sec- 
tions for  2  to  4  minutes  at  most,  washed  off  the  dye  with 
water,  immersed  them  for  5  minutes  or  longer  in  Gram's, 
iodine  solution,  and  then  washed  either  with  alcohol  or  suc- 
cessively with  alcohol  and  clove-oil,  and  mounted  in  Canada 
balsam  ;  or  I  also  subsequently  stained  with  orange.  In  the 
latter  case  I  washed  the  sections,  after  they  came  from  gen- 
tian violet,  but  a  very  short  time  in  alcohol  and  then  placed 
them  in  a  concentrated  aqueous  solution  of  orange,  which 
was  allowed  to  act  a  few  minutes,  then  washed  them  iiD 
alcohol  and  transferred,  in  the  usual  way,  to  Canada  balsam. 

In  good  preparations  where  orange  was  used,  the  nucleoli 
and  the  karyokinetic  figures  from  aster  to  dyaster  were  deep 
red,  the  nuclear  framework  of  resting  nuclei  and  the  spirem 
and  dispirem  were  violet  or  blue,  the  achromatic  nuclear 
figure  and  the  cytoplasm  were  yellow-brown. 

[Gjurasin  has  used  these  methods  with  much  success,  after 
many  others  had  failed,  for  bringing  out  the  karyokinetic 
figures  in  the  asci  of  Peziza^  after  the  material  had  been 
fixed  for  two  days  in  Flemming's  chrom-osmic-acetic  acid.} 


I88  BOTANICAL   MICROTECHNIQUE.  "^TH 

77.  F  u  c  h  s  i  n  .  '^^. 

325.  The  sections  are  first  placed  for  15  minutes  or  longer 
in  a  concentrated  aqueous  solution  of  fuchsin,  then  covered 
with  a  concentrated  solution  of  picric  acid  in  2  parts  water 
and  I  part  alcohol,  in  which  the  stain  becomes  dark  violet, 
then  washed  with  90^  alcohol  as  long  as  any  color  is  given 
off,  then  quickly  rinsed  in  absolute  alcohol,  transferred  to 
xylol  and  finally  to  balsam.  A  very  deep  nuclear  stain  is 
thus  obtained. 

This  stain  may  be  combined  with  methyl  blue  by  placing 
the  sections  for  about  a  quarter  of  an  hour  in  an  aqueous 
solution  of  methyl  blue,  after  washing  out  the  picric  acid 
with  alcohol.  The  methyl  blue  is  then  washed  off  and  the 
sections  are  mounted,  as  usual,  in  balsam. 

O.   Fuchsin-Methyl-green. 

326.  Guignard  (I,  19)  recommends  for  nuclear  staining  an 
aqueous  solution  of  fuchsin  and  methyl  green  which  con- 
tains enough  of  the  two  dyes  to  make  it  appear  deep  violet. 
The  solution  may  be  very  feebly  acidified  with  acetic  acid. 
It  very  quickly  stains  the  nucleus  blue-green  and  the  proto- 
plasm bright  red.  In  case  of  overstaining,  it  may  be  washed 
out  with  water. 

t.   Fuchsin-Iodine-green. 

327.  According  to  Strasburger  (I,  575),  a  mixture  of  fuch- 
sin and  iodine  green,  proposed  by  Babes,  is  very  useful  for 
vegetable  objects.  It  is  prepared  by  pouring  a  solution  of 
iodine  green  in  50^  alcohol  into  an  open  dish  and  adding 
fuchsin,  also  dissolved  in  50^  alcohol,  until  the  fluid  takes  a 
markedly  violet  color.  The  sections  to  be  stained  remain 
about  a  minute  in  this  solution  and  are  then  transferred  to 
glycerine. 

c.  Simultaneous  Fixing   and   Staining. 
a.  Methy  1-green-Acetic-acid. 

328.  To  obtain  a  rapid  staining  of  the  nuclei  with  living 
objects,  the  solution  of  methyl  green  in   \i  acetic  acid,  re- 


I 


SPECIAL    METHODS.  1 89 

commended  especially  by  Strasburger,  may  often  be  used 
with  good  results.  But  very  weak  solutions  of  the  dye  must 
generally  be  used.  Examination  may  be  made  in  the  stain 
itself  or  in  glycerine.  The  preservation  of  preparations 
stained  with  methyl  green  is  not  possible. 


ft.    Picro-nigrosin. 

329.  The  solution  of  nigrosin  in  a  concentrated  aqueous 
solution  of  picric  acid,  recommended  by  Pfitzer  (I),  may  be 
used  with  good  results  for  simultaneous  fixing  and  staining, 
•especially  with  algae.  In  order  to  remove  the  chlorophyll 
at  the  same  time,  a  solution  of  nigrosin  and  picric  acid 
in  96^  alcohol  may  be  used.  At  least  24  hours'  time  is 
usually  necessary  for  good  staining.  The  preparations  are 
then  washed  in  glycerin  and  may  be  preserved  in  glycerine- 
gelatine.  But  the  stain  comes  out  much  more  finely  in 
balsam  or  the  like.    It  is  preserved  well  in  either  medium. 


d.    Staining   intra   vitam. 

330.  The  staining  of  living  nuclei  was  first  accomplished 
in  the  higher  plants  by  Campbell  (I)  by  means  of  dahlia^ 
methyl  violet,  and  mauve'in.  The  author  mentioned  placed 
pieces  of  the  objects  to  be  studied  in  a  .001^  to  .002^  solu- 
tion of  one  of  these  dyes,  and  usually  left  them  there  for 
several  hours.  The  stamen-hairs  of  Tradescantia  virginica 
are  recommended  as  especially  adapted  for  these  studies, 
and  Campbell  succeeded  in  staining  their  nuclei  while  in 
process  of  division.  But  these  stainings  are  usually  pretty 
faint  and  always  much  less  differentiated  than  good  stain- 
ings of  fixed  nuclei.  This  staining  intra  vitam  has  not  led 
to  any  important  results  concerning  the  morphology  of  the 
nucleus. 


190  BOTANICAL   MICROTECHNIQUE. 


II.   The  Resting  Nucleus  and  its  Constituettts, 

a.    Recognition   of  the   Resting   Nucleus. 

331.  If  one  is  concerned  simply  with  the  recognition  of 
resting  nuclei  in  an  organ  or  a  tissue  system,  this  should  no 
longer  present  serious  difficulties  in  the  Phanerogams,  the 
Pteridophytes,  or  the  Mosses.  Especially  by  fixing  with 
chrom-osmic-acetic  acid  (§  309)  or  with  chromic-acid-plati- 
num-chloride (§  312),  and  staining  according  to  one  of  the 
methods  described  in  §§  320  to  325,  this  object  may  always 
be  realized  certainly  and  without  great  trouble.  By  stain- 
ing in  mass  with  haematoxylin  (§  314)  I  have  also  always  ob- 
tained very  sharp  nuclear  staining  at  a  little  distance  from 
the  surface.  Very  instructive  preparations  may  also  be  usu- 
ally obtained  from  algae  and  fungi  by  the  use  of  the  same 
methods.  For  most  algae  1%  chromic  acid  (§  305)  is  an  ex- 
cellent fixing  medium,  and  good  stainings  may  also  be  ob- 
tained by  the  picro-nigrosin  method  (§  329). 

332.  To  stain  the  nucleus  in  the  starch-filled  oospheres  of 
Nitella,  Overton  (II,  35)  used  a  mixture  of  potassium  f err 0- 
cyanide  and  hydrochloric  acid,  diluted  with  8-10  times  its 
bulk  of  water.  The  starch  was  then  converted  into  sufjar 
by  the  hydrochloric  acid  and,  at  the  same  time,  the  Berlin 
blue  produced  by  the  decomposition  of  the  ferrocyanide 
stained  the  nucleus.  This  author  recommends  chloral  hy- 
drate for  clearing. 

333-  Special  difficulties  are  sometimes  due  to  the  cuticu- 
larized  or  slightly  pervious  membranes  which  often  invest 
reproductive  organs.  In  such  cases  the  subsequent  staining 
of  microtome  sections  will  give  the  best  results. 

334.  If  one  wishes,  in  especially  difficult  cases,  as  in  fol- 
lowing the  development  of  spermatozoids,  to  obtain  a  very 
sharp  differentiation  of  nucleus  and  cytoplasm,  double  stain- 
ing may  be  used  with  good  results.  Guignard  (I)  used  for 
this  purpose,  with  material  fixed  with  osmic  acid  or  with 
alcohol,  the  staining  method  with  fuchsin  and  methyl  green 
described  in  §  326. 


SPECIAL   METHODS,  I9I 

b.   The   Constituents  of  the  Resting  Nucleus. 

335.  If  we  examine  more  closely  the  various  constituents  of 
the  nucleus,  the  nucleolus  presents  the  part  which  stains  most 
deeply  with  most  staining  methods.  In  many  objects,  after 
the  use  of  the  above-described  methods  and  very  thorough 
washing,  only  the  nucleoli  appear  very  deeply  stained  in  the 
resting  nuclei.  On  the  other  hand,  the  so-called  chromatin 
spheres  of  the  nuclear  framework  are  distinguished  by 
marked  staining  power,  and  great  differences  in  this  respect 
occur  in  different  plants  and  tissues,  as  well  as  in  similar  cells 
of  different  ages ;  so  that  it  often  happens  that  only  the 
nucleoli  are  stained  in  the  younger  parts,  and  only  the 
nuclear  framework  in  the  older  ones,  by  the  same  method. 
The  cause  of  these  differences  cannot  yet  be  stated ;  but  it 
is  not  improbable  that  bodies  of  various  sorts  are  contained 
in  the  so-called  nuclear  framework. 

336.  The  most  certain  distinction  between  chromatin- 
globules  and  nucleoli  may  be  reached  by  means  of  the  Her- 
mann-Flemming  safranin-gentian-violet  methods  (cf.  §§  322- 
324),  which  give  a  beautiful  red  color  to  the  nucleoli  and  a 
violet  blue  to  the  chromatin-globules,  especially  with  material 
fixed  with  chrom-osmic-acetic  acid  (§  309)  or  with  platinum- 
chloride-osmic-acetic  acid  (§  313).  Whether  these  methods 
give  as  clear  results  in  all  cases  must  be  determined  by 
further  studies. 

336a.  The  general  distribution  of  erythrophiloiis  and  cy- 
anophilous  constituents  of  the  nucleus  was  first  recognized 
by  Auerbach  in  animal  cells.  These  studies  have  lately 
been  extended  to  plants  by  Rosen  (I),  who  recommends  the 
following  methods  : 

1.  Acid  Fuchsin-M ethylene  Blue. — The  sections  are  first 
stained  with  Altmann's  acid  fuchsin  (cf.  §  345),  then  washed 
successively  with  picric-acid-alcohol  and  with  water,  then 
stained  with  methylene  blue,  soaked  in  alcohol,  and  mounted 
in  balsam. 

2.  Fuchsin  and  Methylene  Blue, — First  stain  with  an  aque- 


192  BOTANICAL   MICROTECHNIQUE. 

ous  .1^  solution  of  fuchsin,  then  wash  with  water,  stain 
a^ain  with  .2^  aqueous  solution  of  methylene  blue,  and 
finally  wash  with  alcohol  or  with  a  mixture  of  three  parts 
xylol  and  one  part  alcohol. 

3.  Acid  Fuchsin  and  Methylene  Blue. — The  microtome  sec-j 
tions  fastened  to  the  slide  are  stained  for  half  an  hour  in  ajl 
.\i   aqueous  solution   of  acid   fuchsin,  then   quickly  rinsed   ' 
with  water  and  treated   for  \-\   minute  with  a  .2^  aqueous 
solution  of  methylene  blue.     The  superfluous  stain  is  then 
removed  with  alcohol  and  the  preparation,  as  soon  as  it  is 
air-dry,  is  extracted  with  clove-oil,  in  which  it  may  be  left 
from  6  to  24  hours.     The  clove-oil  is  then  washed  out  with 
alcohol  or  xylol-alcohol,  and  the  preparation  finally  mounted 
in   balsam.     This   method   has   also   been   used   by   Schott 
lander  (I).     He  states,  however,  that  the  time  of  exposur 
to  the  methylene  blue  must  be  much  varied  (between  a  fe 
seconds  and  two  minutes). 

337.  In  many  cases  digestive  fluids  may  be  used  for  th 
recognition  of  the  chromatin-globules.      These  are  not  at 
tacked  by  pepsin,  but  are  quickly  dissolved  by  trypsin  (cf, 

§§  232-4). 

338.  I  will  remark  here  that  Altmann  (III)  has  observe 
a  granula-structure  in  the  resting  nucleus,  which  canno 
be  further  discussed  here,  since  he  has  not  published  th 
methods  used  by  him. 

339.  The    nuclear   membrane   is   also    slightly  capable  o 
staining.     According  to  Fr.  Schwarz  (I,  123),  it  is  best  made 
visible  by  a  205^  solution  of  common  salt  or  of  mono-potasJ 
sium  sulphate,  or  by  a  concentrated  solution  of  potassium 
bichromate  or  the  mixture  of   potassium  ferrocyanide  an 
acetic  acid  mentioned  in  §  238. 

III.    TJie  Kary akinetic  Figures. 

340.  In  nuclei  in  process  of  indirect  or  karyokinetic  divis^ 
ion  there  may  be  distinguished  a  chromatic  and  an  achro- 
matic  figure.     The   former  generally  consists  of  a  nuclear 


SPECIAL   METHODS.  1 93 

thread  composed  of  globules  or  disks  arranged  in  series 
(chromatin-globules),  which  first  falls  into  segments,  each  of 
which  is  then  split  lengthwise.  The  half-segments  thus 
produced  then  separate  so  that  one  of  each  pair  goes  to 
each  daughter-nucleus.  The  achromatic  figure  consists,  on 
the  other  hand,  of  a  number  of  very  delicate  threads  which 
are  usually  arranged  in  the  form  of  a  spindle,  so  that  they 
are  called  the  nuclear  spindle  (cf.  Fig.  36). 

341.  The  chromatic  figure  has,  as  the  name  indicates,  a 
high  staining  capacity,  and  is  also  usually  much  more  deeply 
stained  than  the  nuclear  framework  of  the  resting  nucleus. 
It  agrees  more  with  the  nucleoli  in  its  behavior  with  stain- 
ing media,  although  it  does  not  in  any  case  originate  from 
them. 

In  most  cases,  a  very  deep  staining  of  the  chromatic 
nuclear  figure  is  obtained  by  fixing  with  the  concentrated 
Flemming's  chrom-osmic-acetic  acid  mixture  (cf.  §  309)  and 
staining  with  aniline-water-safranin  (§  320).  In  consequence 
of  their  deeper  staining,  the  dividing  nuclei  are  easily  dis- 
tinguished from  the  resting  nuclei,  even  with  pretty  low 
powers.  Besides  the  above,  chrom.ic-acid-platinum-chloride 
(§  312)  and  chrom-formic  acid  (§  307)  are  especially  useful 
for  fixing,  and  Gram's  method  (§  321)  and  the  fuchsin-picric- 
acid  method  (§  325),  for  staining  the  chromatic  figure.  In 
most  cases  very  good  preparations  of  the  karyokinetic  fig- 
ures are  obtained  by  fixing  with  alcohol  (§  300)  or  alcoholic 
corrosive  sublimate  (§  310)  or  picric  acid  (§  303)  and  staining 
according  to  one  of  the  methods  mentioned,  or  with  one  of 
the  numerous  haematoxylin  (§§  314-317)  or  carmine  solu- 
tions (§§  318,  319). 

I  will  remark  here  that  often  the  karyokinetic  figures  may 
be  made  clearly  visible  without  previous  fixing  by  placing 
sections  of  living  tissues  directly  in  a  concentrated  aqueous 
solution  of  chloral  hydrate,  m  which  usually  only  the  nucleus 
is  left,  of  all  the  cell-contents,  and  the  chromatic  figures 
of  dividing  nuclei  come  out  with  especial  sharpness.  In 
this  way  I  have  obtained  very  instructive  preparations  from 
young  fern-fronds. 


J 94  BOTANICAL   MICROTECHNIQUE.  ^^B 

342.  On  the  other  hand,  it  is  in  most  cases  difficult  to 
make  the  so-called  achrotnatic  figure  clearly  visible.  It  is, 
to  be  sure,  capable  of  staining  to  a  certain  degree,  especially 
when  haematoxylin  is  used,  or  on  double  staining  with 
fuchsin  and  methyl  blue  (§  325),  when  the  chromatic  figure 
is  colored  red,  the  achromatic,  blue.  But,  even  in  these 
preparations,  it  is  difficult  to  clearly  distinguish  the  separate 
threads  of  which  the  achromatic  spindle  is  made  up ;  and  in 
difficult  cases,  as  in  many  fungi,  it  is  usually  impossible  to 
bring  out  this  figure  in  this  way,  A  preliminary  treatment 
with  reagents  will  better  produce  this  result. 

[Strasburger  recommends  fuming  hydrochloric  acid  for 
bringing  out  the  structure  of  the  "  kinoplasm,"  which  in- 
cludes the  achromatic  figure.] 

How  far  the  methods  lately  recommended  by  Flemming 
and  Hermann  for  animal  objects  (cf.  §§  322-324)  will  prove 
of  value  in  these  cases  must  be  determined  by  further  re- 
searches (compare  also  §  348  a-e). 


IV.    The  Inclusions  of  the  Nucleus  {Protein  Crystalloids), 

343.  As  recent  investigations  have  shown,  protein  crystal- 
loids are  pretty  widely  distributed  in  the  nuclei  of  the  Pteri 
dophytes  and  Angiosperms  (cf.  Zimmermann  H,  54,  and 
in,  112);  but  they  do  not  belong  to  their  constant  con- 
stituents, and  it  therefore  seems  to  me  better  to  treat  them 
-as  inclusions  of  the  nucleus,  like  the  starch-grains  and 
crystalloids  contained  in  the  chromatophores.  No  other 
heterogeneous  inclusions  have  yet  been  recognized  in  the 
nucleus. 

344.  In  the  recognition  of  protein  crystalloids  their  regu- 
lar crystalline  form  is  in  many  cases  an  aid.  Thus  the  crys- 
talline structure  of  the  crystalloids  from  the  leaves  of  Melam- 
Pyrum  arvense  and  Ca7idollca  adnata,  shown  in  Fig.  34,  2  and 
4,  can  hardly  be  questioned.  Besides,  one  very  often  finds 
also  needle-like  or  rod-like  crystalloids,  as  in  the  ovules  of 


I 


SPECIAL   METHODS. 


195 


the  ovary-wall  of  Campanula  trache- 
lium.  2,  nucleus  from  the  spongy- 
parenchyma  of  the  leaf  of  Melam- 
pyrum  arvense.  3,  nucleus  from  the 
epidermis  of  the  leaf  of  Lophospermuvt 
scandens.  4,  nuclei  from  the  palisade- 
parenchyma  of  the  leaf  of  Candollea 
adnata.  5,  nuclei  from  the  wall  of  an 
almost  ripe  fruit  of  Alectorolophus 
major.  6.  nuclei  from  the  ovary  of 
Mimulus  Tillingi.  «,  nucleolus.  p, 
protein   crystalloids. 


Mimulus   Tillingi  (Fig.  34,  6),  or  variously  curved  forms,  as 

in    the    wall    of    the    ovary    of 

Campanula     trachelium      (Fig. 

34,  i).      Finally,  bodies  of  the 

same     chemical     relations    are 

widely  distributed,  which  vary 

httle  or  none  from  the  globular 

form,  as  for  example  in  the  leaf 

of  Lophospermiim  scandens  (Fig. 

34,  3).  In  such  cases  the  certain 

proof  of  their  crystalloid  nature,     f,c.  34-1,  nuclei  from  the  epidermis  of 

and    especially   the.   distinction 

between  them  and  the  nucleoli, 

is   not   possible   by   the   simple 

examination  of  living  material. 

But    this    distinction    may    be 

easily  and  certainly  made  by  the 

aid  of  suitable  staining  methods.  These  leave  any  doubt  only 

as  to  whether  all  those  bodies  which  correspond  with  the 

undoubted  crystalloids  are  really  to  be  regarded  as  identical 

with  them  in  substance ;  but  it  may  be  considered  as  certain 

that  these  are  not  identical  with  any  other  knoiim  inclusions 

of  the  nucleus. 

For  the  recognition  of  crystalloids  one  may  best  fix  the 
material  with  a  concentrated  alcoholic  solution  of  corrosive 
sublimate  (§  3 10)  and  stain  with  acid  fuchsin,  or  use  a  double 
staining  of  acid  fuchsin  and  haematoxylin.  As  to  the  stain- 
ing with  acid  fuchsin,  which  is  also  known  as  "  Fuchsin  S 
after  Weigert,"  this  may  be  conducted  in  various  ways ;  but 
the  three  following  methods  have  proved  best  heretofore 
(cf.  Zimmermann  II,  12  and  55,  and  III,  113). 

a.     Altmann's  Acid  Fuchsin  Staining. 

345.  This  is  chiefly  useful  for  microtome  sections.  These 
are  fastened  to  the  slide  and,  after  the  removal  of  the  par- 
afifine,  are  covered  with  a  solution  of  20  grams  of  acid  fuch- 
sin in  100  ccm.  of  aniline-water,*  and  then  warmed  until  the 


*  This  solution  keeps  well  and  only  needs  to  be  filtered  occasionally. 


196  BOTANICAL   MICROTECHNIQUE. 

under  side  of  the  slide  is  very  hot  to  the  touch.  BoiHng  the 
solution  is  to  be  avoided,  though  its  complete  drying  up 
does  not  injure  the  staining.  When  the  solution  has  acted 
from  two  to  five  minutes,  or  longer,  it  is  rinsed  off  with  a 
mixture  of  one  part  alcoholic  picric  acid  solution  and  two 
parts  water,  and  the  washing  with  this  solution  is,  in  general^ 
continued  until  the  sections  no  longer  give  off  any  visible 
color.  But  in  many  cases  various  degrees  of  staining  may 
be  obtained  by  stopping  the  washing  earlier  or  later.  The 
picric  acid  is  finally  removed  with  alcohol,  and  the  prepara- 
tion mounted  in  Canada  balsam  in  the  usual  way. 

It  may  be  observed  that,  according  to  Altmann's  direc- 
tions, the  sections  should  be  gently  warmed  again  in  picric 
acid  solution  after  being  washed  with  it,  in  order  to  obtain 
good  differentiation.  But  with  vegetable  objects  I  have  in 
most  cases  obtained  no  good  results  from  this  warming  with 
picric  acid,  while  otherwise  this  method  has  repeatedly  done 
me  good  service. 

With  this  method  a  very  deep  staining  of  the  nuclear  crys- 
talloids is  always  obtained  ;  but  it  is  inferior  to  the  two  fol- 
lowing methods  in  that,  especially  in  young  cells,  at  least 
when  fixed  with  sublimate,  the  nucleolus  is  also  pretty  deep- 
ly stained. 

b.    Acid    Fuchsin  Method  B. 

346.  The  second  method,  which  I  have  called  "  acid  fuch- 
sin method  B"  (cf.  Zimmermann  II,  14),  is  adapted  as  well 
for  free-hand  sections  as  for  those  from  the  microtome.  The 
well-washed  sections  are  placed  first  in  a  .2^  solution  of  acid 
fuchsin  in  distilled  water,  to  which  a  little  camphor  is  added 
to  make  it  keep  better.  They  remain  in  this  solution  at 
least  several  hours,  best  24  hours  or  longer.'^  They  are  then 
washed  as  quickly  as  possible  in  running  water  (cf.  §  39). 
The  time  necessary  for  this  differs  much  in  different  cases, 
and  varies  between  a  few  minutes  and  several  hours.    But  it 

*  If  many  sections  are  to  be  stained  at  the  same  lime  in  this  way,  the  ves- 
sel described  in  §  37  may  be  used. 


SPECIAL   METHODS.  \<^T 

may  be  determined  easily  by  a  couple  of  trials.  After  wash- 
ing, the  preparations  are  transferred  in  the  usual  way  tO' 
Canada  balsam. 

By  the  use  of  this  method  I  have  obtained  a  very  good 
staining  of  the  nuclear  crystalloids  in  both  free  hand  and 
microtome  sections.  They  are  always  deeply  stained,  even 
if  all  the  other  constituents  of  the  nuclei,  even  the  nucleoli^, 
have  been  decolorized  long  before. 

c.     The  Acid-fuchsin-Potassium-bichromate  Method. 

347.  The  third  method,  which  may  perhaps  be  briefly 
called  the  "acid  fuchsin  method  C,"  agrees  almost  complete- 
ly with  Altmann's  method  (§  345)  in  that  here  also  the 
microtome  sections  are  warmed  with  a  concentrated  solutiorfc 
of  acid  fuchsin.  But  for  washing  a  warmed  solution  of 
potassium  bichromate  is  used  instead  of  picric  acid,  and  the 
result  depends  neither  on  the  concentration  of  the  solution, 
nor  on  the  maintenance  of  a  definite  temperature.  I  used 
mostly  a  concentrated  aqueous  solution  of  the  salt  heated  in 
the  parafifine  oven  to  50°  or  60°  C,  but  even  a  boiling  solution 
may  be  used.  When  the  preparations  are  sufficiently  washed, 
which  can  usually  be  readily  recognized  after  a  little  practice,, 
but  may  be  determined  by  a  few  trials,  the  potassium  bichro- 
mate is  quickly  washed  with  water  and  then  the  preparation 
is  transferred  to  balsam  in  the  usual  way. 

By  the  use  of  this  method  I  obtained  always  very  clear 
staining  of  the  nuclear  crystalloids.  These  remained  still 
deeply  stained  when  the  color  had  long  been  washed  out  of 
the  nucleolus. 

d.  Double  Staining  with  Acid  Fuchsin  and  Haematoxylin. 

348.  This  method  may  be  used  in  very  different  ways. 
But  I  have  generally  found  it  best  to  first  stain  the  objects- 
in  mass  with  Delafield's  haematoxylin  (§  314),  and  then  to 
stain  microtome  sections  cut  from  them  with  acid  fuchsin  by^ 
the  method  B  (§  346).  Then,  in  one  and  the  same  nucleus 
may  be  seen  the  deep  red-colored  crystalloids  beside  the 
deep  blue-violet  nucleolus,  within  the  violet  nuclear  frame- 


198 


BO  TA  NIC  A  L   MICRO  TECHNIQ  UE. 


work.  This  method  has  also  proven  good  for  following  the 
fate  of  the  crystalloids  during  karyokinesis  (Zimmermann, 
III,  119). 

2.    The   Centrospheres. 

348a.  After  the  presence  of  the  so-called  centrospheres* 
or  attractive  spheres  had  been  recognized  in  animal  cells  by 
various  authors  (cf.  Flemming  I  and  III),  Guignard  (IV)suc- 
■ceeded  in  observing  them  in  various  plant-cells  also,  and  it 
is  now  to  be  regarded  as  not  improbable  that  these  bodies 
-are  constant  constituents  of  the  cell. 

The  centrospheres,  which  have  also  been  called  by  Guig- 
jiard  ''spheres   directrices,'*   consist   of  a  generally  globular 
central  portion   (the  "centrosome"   of  Guignard)  which   is 
surrounded  by  an  unstainable  envelope,  and  occur  usually 
a  pair  in  each  cell  (cf.  Fig.  35  and  36,  a). 
They  appear  to  play  an  important  part, 
especially   in    karyokinesis.     At    least, 
they   form,  after  their  separation,  the 
centres  of  the  radiating  structures  ob- 
served   in   the   cytoplasm  (cf.  Fig.  36, 
II);  and  the  threads  of  the  achromatic 
spindle  (cf.  §  342)  run  together  at  both 
Fig.  35.-Embryo-sac  of  /./-  euds  at  the  attractive  spheres  (cf.  Fig. 

liufti  MartagoH  before  the       ^ttt  ittt\  a.i 

first  nuclear  division,  a,  36,   ill   and  IV).     At  the   samc    time 

centrospheres.  After  Guig-        .   ,       ,  1  •      •  r     1 

nard.  With  the  Splitting  of  the  segments  of  the 

nuclear  threads,  a  division  of  the  centrospheres  occurs,  so 
that  each  daughter-nucleus  has  again  two  attractive  spheres 
{cf.  Fig.  36,  IV),  which  at  first  remain  together,  and  separate 
only  at  the  beginning  of  another  nuclear  division. 

In  the  sexual  act  of  the  Angiosperms,  according  to  Gui- 
gnard's  observations,  the  attractive  spheres  of  the  male 
nucleus  enter  the  egg-cell  at  the  same  time  with  it,  and  the 
spheres  of  male  and  female  origin  fuse  in  pairs  (cf.  Fig.  37). 

*[This  English  equivalent  of  the  term  lately  proposed  by  Strasburger  for 
these  structures  seems,  on  the  whole,  the  most  available  name  for  them. 
The  non-staining  envelope  of  the  centrosome  may  be  termed,  with  Stras- 
fcurger,  the  astrosphere.\ 


SPECIAL    METHODS. 


199^ 


348b.  Guignard  recommends  (IV,  166),  for  bringing  out 
the  centrospheres,  fixing  with  alcohol,  a  70-30^  alcoholic 
solution  of  corrosive  sublimate  or  picric  acid,  a  li  aqueous 
solution  of  corrosive  sublimate,  a  saturated  aqueous  solution 
of  picric  acid,  or  a  .5^  solution  of  chromic  acid.     He  has 


Fig.  ■>,().— Lilium  Marta^on.  I,  tip  of  the  embryo-sa*;  II,  the  same,  later  stage;  III 
and  IV,  older  karyokmeiic  figures  from  the  same  source;  a,  centrospheres.  After 
Guignard. 

also  used  the  vapor  of  osmic  acid,  but  allows  it  to  act  only 
a  short  time,  in  order  not  to  lessen  the  staining  capacity  of 
the  objects,  and  then  places  them  in   Flemming's  solutioa 
(§  309)  for  half  an  hour  to  an  hour,  and 
then  in  alcohol. 

For  staining  the  attractive  spheres 
Guignard  uses  especially  hsematoxylin  ; 
but  he  first  treats  the  sections  hardened 
with  alcohol  with  a  lO'fc  solution  of  zinc 
sulphate  or  ammonia  alum.  He  has  yig. 
also  treated   the   preparations   success- 


-Nuclei  from  the  fer- 
tilized egg-cell  of  Liliuvt 
Martagon  during  their  fu- 

1  •.■!  jM     ^  1     4.: ^C      sion.     w,    male,    ^,    female 

IVely  With  a  dilute    aqueous    solution    01       nucleus;    «,  centrospheres. 

orseillin  and  eosin-haematoxylin.*     '^^^        '^"^    uignar  . 


The 


*  This  probably  means  the  eosin-haematoxylin  mixture  recommended  by 
Renault.  It  is  prepared,  according  to  Gierke  (I,  86),  by  mixing  equal 
parts  of  glycerine,  containing  common  salt  and  saturated  with  eosin,  and  a 
saturated  solution  of  potash  alum  in  glycerine.  This  mixture  is  filtered 
and  then  an  alcoholic  solution  of  haematoxylin  or  Delafield's  haematoxylin 
(§  314)  is  added. 


:20O  BOTANICAL   MICROTECHNIQUE. 

interior  of  the  centrosphere  is  stained  deep  red  by  tliis 
treatment. 

For  moiniting  such  preparations,  Guignard  recommends 
especially  glycerine-gelatine  and  a  lO^  solution  of  chloral 
hydrate  thickened  with  gelatine.  The  latter  has  the  advan- 
tage of  clearing  the  preparations,  but  gradually  destroys 
most  dyes. 

348c.  Guignard  has  succeeded  in  bringing  out  the  attrac- 
tive spheres  especially  in  various  sexual  cells.  In  the  grow- 
ing stamen-hairs  of  Tradescantia  he  also  succeeded  by 
treating  them  successively  with  osmic  acid  vapor,  Flem- 
ming's  chrom-osmic-acetic  acid  mixture,  and  alcohol,  and 
then  staining  with  a  mixture  of  fuchsin  and  methyl  green. 
If  this  mixture  is  rightly  prepared,  the  centrospheres  are 
-colored  bright  red  in  the  pale  red  protoplasm. 

348d.  Hermann  (II,  583)  has  recently  used  the  following 
method  for  making  visible  the  centrospheres  and  the  radi- 
ating structures  around  them,  in  animal  cells.  The  ob- 
jects, fixed  with  platinum-chloride-osmic-acetic  acid  and  then 
reduced  with  wood-spirit  in  the  manner  described  in  §  313, 
are  placed  whole  in  the  dark  in  a  haematoxylin  solution  con- 
taining one  part  haematoxylin,  70  parts  alcohol,  and  30  parts 
water.  They  remain  in  this  solution  12  to  18  hours,  are 
then  treated  for  the  same  time,  also  in  the  dark,  with  70^ 
alcohol,  and  are  then  imbedded  and  sectioned  with  the 
microtome.  The  sections  are  then  extracted  with  a  solu- 
tion o{  potassium  permanganate  so  dilute  that  it  has  a  bright 
rose  color,  until  they  have  an  ochre-colored  appearance. 
After  rapid  rinsing  in  water,  the  manganese  peroxide  is  dis- 
solved out  with  a  solution  of  one  part  oxalic  acid  and  one 
part  potassium  sulphate  to  1000  to  2000  parts  of  water,  and 
the  sections  are  then  stained  for  three  to  five  minutes  with 
safranin.  The  attractive  spheres  and  the  structures  sur- 
rounding them  appear  deeply  blackened,  while  the  nuclear 
elements  have  a  bright  red  color. 

How  far  this  method  can  be  used  with  success  for  plant- 
cells  remains  to  be  shown.  But  I  will  remark  that  the 
methods  used  by  Flemming  on  animal  cells  with  the  best 


SPECIAL   METHODS.  20I 

results  (cf.  §  323)  are  poorly  suited  to  plant-cells,  according 
to  Guignard  (IV,  167).  I  have  obtained,  also,  in  some  not 
very  extended  experiments  with  Hermann's  methods,  no 
staining  of  the  centrospheres,  while  the  spindle-threads  of 
such  preparations  stood  out  very  sharply,  especially  after 
staining  with  gentian  violet. 

3.  The  Chromatophores  and  their  Inclusions. 

349.  Under  the  name  chromatophores  are  commonly  in- 
cluded at  present  three  different  kinds  of  bodies ;  the  green 
chlorophyll-bodies,  chloroplasts,  and  the  corresponding  bod- 
ies in  the  algae  which  are  not  green,  the  mostly  yellow  or 
red  bodies  which  carry  coloring  matters,  chromoplasts,  oc- 
curring especially  in  the  bright-colored  parts  of  flowers  and 
fruits,  and  the  colorless  leucoplasts^  which  are  found  chiefly 
in  subterranean  and  young  parts  of  plants.  The  grouping 
of  these  different  bodies  together  is  justified,  aside  from 
their  chemical  similarity,  by  the  fact,  recognized  especially 
by  Schimper,  that  they  stand  in  genetic  relations  with  each 
other,  and  may  pass  over  into  each  other  in  the  most  vari- 
ous ways 

I.  Methods  of  Investigation. 

350.  The  study  of  chromatophores  has  been  conducted 
chiefly  in  the  living  cell.  Of  course  this  is  only  possible  in 
sections  which  are  at  least  several  cell-layers  in  thickness; 
and  the  most  rapid  preparation  possible  is  necessary,  since 
chromatophores  are  very  sensitive  to  the  most  varied  harm- 
ful influences.  Since  most  cells  also  die  very  quickly  in 
pure  water,  and  the  chromatophores  especially  suffer  pro- 
found structural  changes  in  this  medium,  it  is  advantageous 
to  use  a  dilute  solution  of  salt  or  sugar  as  a  medium  for 
their  study.  I  have  used  with  good  results  a  5^  solution  of 
sugar,  with  which  I  injected  the  tissues  to  remove  the  air 
from  the  intercellular  spaces,  which  may  usually  be  easily 
done  by  means  of  a  filter  pump  (cf.  also  §  5). 

351.  In  difficult  cases  one  must  have  recourse  to  staining 
methods.     I  have  found  a  concentrated  alcoholic  solution  of 


202 


BO  TANICA L   MICRO  TECHNIQ UE. 


corrosive  sublimate  well  adapted  for  fixing  (cf.  §  310);  and 
a  concentrated  alcoholic  picric  acid  solution  often  does, 
well. 

According  to  ray  own  most  recent  experiments,  a  saturated 
solution  of  picric  acid  and  corrosive  sublimate  in  absolute 
alcohol  seems  to  be  best  for  fixing  chromatophores.  I  allow 
it  to  act  about  24  hours  on  the  objects  to  be  fixed  and  wash 
it  out  with  running  water.  The  use  of  an  iodine  solution 
for  the  removal  of  the  sublimate  seems  unnecessary  here,, 
as  I  have  seen  none  of  the  well-known  sublimate  needles  in 
my  preparations. 

Krasser  (II,  4)  recommends  the  use  of  a  \ic  alcoholic  so- 
lution of  salicylic  aldehyde  for  fixing  chromatophores.  He 
lets  it  act  for  24  to  48  hours  on  small  pieces  of  tissue.  After 
hardening  in  alcohol,  the  sections  may  be  mounted  in  gly- 
cerine, glycerine-gelatine,  or  balsam.  If  in  the  latter,  the 
clearing  in  clove-oil  must  be  made  as  brief  as  possible. 

352.  Schimper  used  haematoxylin  and  gentian  violet  for 
staining  chromatophores;  but  I  have  found  iodine  green» 
fuchsin,  and  acid  fuchsin  better  (cf.  Zimmermann  V,  6). 

Staining  with  acid  ftichsin  is  best  accomplished  by  one  of 
the  three  methods  described  in  §§  345  to  347.  It  is  easy  to 
make  clearly  visible  the  relatively  small  leucoplasts  on  each 
starch-grain  in  the  outer  layers  of  a  ripe  potato,  by  the  aid 

of  method  B  (cf.  Fig.  38,  /). 

353-  Iodine  green  is  used  in 
concentrated  aqueous  solution 
and  is  either  allowed  to  act 
for  only  a  short  time  {^  to  a 
few  minutes)  on  microtome 
sections,  which  are  then 
washed  with  water  and  exam- 
ined in  glycerine ;  or  it  is  al- 
lowed to  act  longer  and  the 

a  few  layers  removed  from  the  cork.    After    sectionS  are  thcn    placed  in    a 
Iixinjjr  with  sublimate-alcohol  and  staining  ^ 

•euco-  solution  prepared  by  mixing 

two  parts  of  common  ammo- 

In  this  the  sections  were  left 


Fig.  38. — Cell-contents  from  a  parenchyma 
celf  of  a  tuber  of  Solanum  tuberosum,  but 


with  acid  fuchsin  (Method  B).    /, 
plasts ;  J,  starch-grains  ;  z,   nucleus 
crystalloid. 

nia  with  98  parts  of  water. 


SPECIAL   METHODS. 


203 


from  a  few  minutes  to  several  hours,  according  to  the  depth 
of  the  staining  and  the  character  of  the  preparation.  The 
examination  may  be  made  in  glycerine.  Such  preparations 
keep  only  a  very  short  time,  while  very  permanent  prepara- 
tions may  be  made  by  transferring  to  Canada  balsam  even 
sections  stained  with  iodine  green.  In  this  transfer  alcohol 
must  be  wholly  avoided,  since  it  decolorizes  the  chromato- 
phores.  Phenol  and  aniline  also  are  not  suited  for  this  use. 
Therefore  I  simply  allowed  the  sections  to  dry,  after  wash- 
ing them  with  water,  and  then  treated  them  with  xylol,  and 
finally  added  xylol-balsam. 

354.  For  staining  with /7/<;//5/;/,  I  have  used  the  ammonia 
fuchsin  mentioned  in  §  271.  I  let  it  act  only  a  short  time 
on  the  sections,  until  they  begin  to  become  red.  Then  I 
wash  it  out  with  water,  and  examine  the  sections  in  glycer- 
ine or  transfer  them  in  the  above  described  manner,  by  dry- 
ing, to  balsam.  Alcohol  decolorizes  the  chromatophores  in 
this  case,  also. 


II.    The  Finer  Structure  of  the  Chromatophores. 

355.  Opinions  are  at  present  divided  as  to  the  intimate 
structure  of  the  chromatophores  (cf.  Zimmermann  I,  56  and 
Bredow  I,  380,  for  the  early  literature).     It  is  only  as  to  the. 


P    I 


jr 


M 


pi         A 


@ 


Fig.  39. — I,  chromoplasts  from  the  flower  of  Neottia  nidus-avis  \  /,  protein  crystalloids  ; 
fy  pigment-crystals.  II,  the  same,  from  the  root  of  Daucus  Carota.  Ill,  the  same, 
from  the  fruit  of  Sorbus  aucuparia.    s,  starch-grains.— After  Schimper. 


chromoplasts  that  it  may  be  regarded  as  settled  that  the 
pigment  occurs  partly  in  crystalline,  partly  in  amorphous 
form. 

356.  In  the  former  case,  it  forms  more    or  less   regular 


204  BOTANICAL   MICROTECHNIQUE. 

rhombic  plates  or  peculiar  cylindrically  curved  bodies,  as, 
for  example,  in  the  parenchyma  of  the  carrot  (cf.  Fig.  39, 
II);  or  it  occurs  in  the  form  of  delicate  needles  which  are 
imbedded  in  the  colorless  stroma  in  small  numbers,  as  in 
Neottia  nidus-avis  (Fig.  39,  I),  or  in  large  quantity,  as  in  the 
pericarp  of  Sorhus  auaiparia  (Fig.  39,  III).  All  these  pig- 
ment-crystals, which  consist  of  carotin,  according  to  the 
prevailing  nomenclature,  are  characterized  by  strong  pleo- 
chroism  *),  and  this  peculiarity  has  been  used  by  Schimper 
<III,  94)  in  difficult  cases  for  the  recognition  of  the  crystal- 
line structure  of  the  fine  pigment-needles. 

357.  The  amorphous  pigment  occurs  within  strongly  re- 
fractive globules  {'' grana'')  which  are  imbedded  in  the 
usually  quite  colorless  stroma.  The  flesh- 
colored  fertile  stems  of  Egiiisetuvi  arvensc  con- 
tain  chromatophores  with  especially  large 
grana  (Fig.  40). 

The  study  of  the  crystalline  and  amorphous 
pierments  can,  of  course,    be  carried    on  with 

Fig.  4o.-Chromo-   ^  ^.  .     '  / 

piasts   from  the  entire  certamtv    only  withm  the   hvmpr  cells, 

parenchyma       of  •'  ^     ^  ^    ^    ^ 

the  stem  of  ^y«/-  and,  on  account  of  their  ready  decomposition, 

setum       arvense. 

(X1400.)  tj-je  precautions  described  in  §  350  should  be 

very  strictly  adhered  to. 


III.    The  Incltisions  of  the  Chromatophores. 

358.  The  most  widely  distributed  of  the  inclusions  of  the 
t:hromatophores,  the  starch-grains,  will  be  treated  in  connec- 
tion with  various  related  bodies  in  §  400  and  following  ones. 
Besides  these,  there  have  been  recognized  in  the  chromato- 
phores protein  crystalloids,  leucosomes,  pyrenoids,  and  oil- 
drops,  which  will  now  be  discussed  in  order. 


*This  is  determined  by  observing  the  body  with  one  nicol  prism,  prefer- 
ably the  polarizer,  and  turning  either  the  nicol  or  the  crystal.  PleochroYc 
objects  then  appear  quite  colorless  in  one  position,  or  at  least  very  light 
colored,  and,  after  rotation  through  90°,  more  deeply  colored  than  without 
the  use  of  the  nicol. 


SPECIAL   METHODS. 


205 


a.  Protein  Crystalloids. 

359.  The  protein  crystalloids  observed  in  the  chromato- 
phores,  especially  by  Schimper  (III)  and  A.  Meyer  (II),  often 
form  elongated  prisms  (cf.  Fig.  41,  2)  or  needles  (Fig.  41,  i 
and  Fig.  39,  I,/)  ;  but  they  are  not  rarely  more  octahedral 
or  more  or  less  irregular  (cf.  Fig.  41,  i,  3,  and  4). 

With  the  exception  of  those  of 
Caniia,  they  are  soluble  in  water, 
but  are  fixed  by  fixing  media  for 
proteids.  I  have  found  the  same 
methods  used  for  the  study  of  the 
nuclear  crystalloids  (cf.  §§  345  to 
347)  well  suited  to  their  investi- 
gation. Fixing  with  an  alcoholic 
solution    of    corrosive   sublimate 

,      ^    .     .  •    .  •  1   r       1      •       1         ^'<^-  4I-— I,  leucoplasts   from  a  youncr 

and  staining  with  acid  tuchsin  by     shoot  of  canna  Warszewiczn  wUh 

,1^1  111  crystalline    needles    and    octahedral 

method  C  has  proved  the  best 
treatment.  In  this  way  it  is 
pretty  easy  to  obtain  prepara- 
tions in  which  the  stroma  of  the 
chromatophores  is  quite  colorless, 
while  the  crj'stalloids  are  colored  deep  red 


crystals;  2.  chloroplasts  from  the 
epidermis  of  the  petiole  of  Hedera  sp.; 
3,  chloroplasts  from  the  palisade-pa- 
renchyma of  the  leaf  of  Convolvulus 
tricolor  \  4,  chloroplasts  from  the 
palisade-parenchyma  of  the  leaf  of 
Achyrantkes  Verschaffelti  ;  k,  pro- 
tein crystalloids;  j,  starch-grains. 
I,  after  Schimper. 


b.  Leucosomes. 


360.  Within  the  leucoplasts  of  the  epidermis  of  various 
species  of  Tradescantia,  and  in  various  other  plants,  I  have 
found  globular  inclusions  which  I  have  provisionally  termed 
leucosomes  (cf.  Zimmermann  II,  3  and  III,  147).  These 
consist,  at  least  chiefly,  of  protein-like  substance  and  are 
probably  closely  related  chemically  to  the  protein  crystal- 
loids already  described,  with  which  they  also  correspond  in 
their  behavior  with  acid  fuchsin. 

In  favorable  cases,  as,  for  instance,  in  the  epidermis  of  the 
leaves  of  Tradescantia  discolor,  the  leucosomes  may  be  well 
observed  within  the  living  cell,  and  tangential  sections  are 
especially  adapted  to  this  study.     They  occur  here  mostly 


2o6 


BOTANICAL   MICROTECHNIQUE, 


singly  or  in 


KJ^— 


§) 


^ 


twos  or  threes  in  each  leucoplast  (cf.  Fig.  42,  /). 

But,  since  the  leucosomes  are  soluble  in  water 
and  very  sensitive  to  the  most  various  reagents,, 
it  is  better  to  use,  as  a  rule,  fixed  and  stained 
material. 

The  best  method  is  to  fix  sections  from  the 
living  objects  in  a  concentrated  alcoholic  solu- 
tion of  corrosive  sublimate,  and,  after  Avashing^ 
(cf.  §  310),  to  stain  by  the  acid  fuchsin  method 
B.  If  the  washing  is  stopped  at  the  right 
moment,  the  leucosomes  alone  are  deeply  stained. 


Fig.  42— Leuco- 
plasts  from  the 
epidermis  of 
the  upper  sur- 
face of  the  leaf 
of     Tradescan- 

\  tia  discolor.  /, 
leucosomes. 


c.  Pyrenoids. 

361.  The  starch-centres  or  pyrenoids  observed  in  the 
chromatophores  of  various  Algce  and  of  Anthoceros  consist 
of  a  central  portion  of  proteid  and  a  starch-envelope  sur- 
rounding it. 

The  most  essential  part  of  the  pyrenoid,  the  proteid  nu- 
cleus, appears  often  to  possess  a  more  or  less  regular  cry.stalline 
form  (cf.  Fig.  43,  2),  according  to  Schimper's  observations 
(III,  74).  On  the  other  hand,  it  is  certain  that  non-crystal- 
line pyrenoids  also  occur,  as  in  case  of  the  one  shown  in 
Fig.  43,  3,  which  probably  represents  a  stage  in  fission  (cf. 
Zimmermann  I,  48).  Similar  forms  have  lately  been  ob- 
served by  Klebahn  (I,  426)  in  Cosmaruim. 

362.  In  the  presence  of  large  quantities  of  starch,    the 


Fig.  43 

sp-;   _ 

with  acid  fuchsin 
Schimper, 


I,  part  of  a  chromatophore  of  Spirogyra  sp. ;    2,  chromatophores  of  Cladophora 

3,  constricted  pyrenoid  of  Zygnema  sp.  after  fixing  with  picric  acid  and  staining 

/,  pyrenoid  ;  j,  starch-grains  ;  «,  nucleus  ;  «,  nucleolus.     2,  after 


proteid  nucleus  of  the  pyrenoid  is  only  with  difficulty  or  not 
at  all  to  be  recognized  within  the  living  cell.  Then  the  use 
of  suitable  staining  media  is  to  be  recommended.     The  py- 


SPECIAL   METHODS,  20/ 

renoids  behave  with  these  in  about  the  same  way  as  do  the 
crystalloids  of  the  nuclei  and  the  chromatophores.  Very 
■deep  staining  of  them  may  be  obtained  by  fixing  algae  with 
picro-sulphuric  acid  (§  304)  or  an  alcoholic  solution  of  cor- 
rosive sublimate  (§  310),  washing  well,  and  then  leaving  them 
at  least  24  hours  in  a  .2^  solution  of  acid  fiichsin.  After 
cashing  in  water  for  a  quarter  of  an  hour  and  dehydrating 
in  Schulze's  apparatus  (§  16),  the  objects  should  be  trans- 
ferred to  Canada  balsam  by  the  aid  of  Schulze's  settling 
cylinder  (§  21).  In  good  preparations  only  the  pyrenoids 
are  deep  red,  and  even  the  nucleoli  are  quite  colorless. 

363.  A  simultaneous  fixing  and  staining  of  chromato- 
phores and  pyrenoids  may  be  obtained  more  simply  by 
placing  the  algae  in  a  concentrated  solution  of  picric  acid  in 
50^  alcohol,  to  which  a  little  acid  fuchsin  is  added  (about 
five  drops  of  Altmann's  solution,  described  in  §  345,  to  a 
watch-glass  full  of  the  picric  acid  solution).  In  this  they  re- 
main for  two  hours  or  longer,  ancj  are  then  washed  for  a 
quarter  of  an  hour  in  alcohol  (not  previously  in  water)  and 
then  transferred  as  quickly  as  possible  to  balsam  by  the  aid 
of  Schulze's  settling  cylinder  (§  21).  Preparations  thus  ob- 
tained appear  to  keep  well,  while  a  gradual  fading  occurs  in 
those  mounted  in  Vosseler's  turpentine  (§  27). 

363a.  According  to  the  researches  of  Hieronymus  (I,  358), 
the  pyrenoids  of  Dicranochcete  reniformis  appear  to  have  a 
more  complicated  constitution.  This  author  states  that 
they  consist  of  a  protein  crystalloid  and  a  protei'd-like  en- 
velope, while  the  starch-grains  are  formed  quite  independ- 
ently of  the  pyrenoids,  at  any  part  of  the  chromatophore. 

The  crystalloids  are  soluble,  according  to  Hieronymus, 
in  boiling  water  (I),  dilute  and  concentrated  caustic  potash 
solution,  common  salt  solution,  acetic  acid,  and  hydrochloric 
acid.  After  previous  treatment  with  alcohol,  its  solubility 
becomes  less.  After  lying  for  several  days  in  a  mixture  of 
one  volume  of  pepsin-glycerine  and  three  volumes  of  .2^ 
hydrochloric  acid,  the  crystalloids  become  very  transparent, 
Hieronymus  recommends  safranin  for  staining  them. 

The  envelope  is  also  soluble  in  concentrated  and  dilute 


208  BOTANICAL   MICROTECHNIQUE. 

caustic  potash  solution,  as  well  as  in  hydrochloric  acid  of 
various  strengths.  But  it  is  not  attacked  by  the  above- 
mentioned  digesting  fluid,  even  after  lying  in  it  for  days. 
HcBfuatoxylin  and  Congo  red  are  specially  adapted  to- 
staining  the  envelope.  By  the  combination  of  safranin  and 
haematoxylin,  Hieronymus  obtained  preparations  in  which 
the  envelope  was  stained  violet  and  the  crystalloid  deep- 
red. 

d.  Oil-drops. 

364.  Drops  of  an  oil-like  substance  are  pretty  widely 
distributed  within  chromatophores,  as  has  been  shown 
especially  by  A.  Meyer  (II),  and  Schimper  (III,  106).  Ac- 
cording to  A.  Meyer's  researches  (II.  17),  however,  these  do 
not  fully  correspond  in  their  chemical  relations  either  with 
the  true  fatty  oils  or  with  the  ethereal  oils.  It  is  especially 
noteworthy  that  they  are  soluble  even  in  dilute  alcohol,  but 
insoluble  in  acetic  acid.  They  are,  further,  only  very  grad- 
ually browned  by  osmic  acid,  and  are  always  insoluble  in 
water,  but  readily  soluble  in  ether.  They  behave  variously^ 
with  chloral  hydrate.  According  to  all  these  reactions,  the 
bodies  in  question  must  surely  be  nearly  related  to  the 
fatty  oils,  so  much  the  more  that  they  stain  deeply  with 
alcannin  and  cyanin  by  the  use  of  the  methods  previously 
described  (§§  109,  no).  At  all  events,  it  seems  to  me  best 
to  designate  these  bodies  as  oil-drops,  as  has  usually  been 
done  in  the  literature,  so  long  as  no  exact  investigations  of 
them  exist. 

I  can  recommend  as  a  suitable  object  for  the  study  of  the 

reactions  of  these  oil-drops,  the  epidermis  of 

/T\    (flg.        t^^^  1^^^  o^  Agave  amtricana.     This  contains- 

^^y  \)      leucoplasts  which  always  enclose  pretty  large 

/   /"^r"^^      oil-drops,  especially  in  old  leaves  (cf.  Fig.  44). 

\^  In  yellowed  leaves    these  have    usually  run 

Fic.  44.-Leuco-    together  into  a  single  large  drop,  beside  which 

ep^de'rmirof  ^aJ    ^^^^  ^^^^  ^^  ^^^  leucoplast  is  SO  inconspicuous 

""A^^ve    ^aterul.    ^^^^^   it  cannot  usually  be  directly  made  out 

na.  ^.oil-drops,     ^jtj^  certainty,  by  direct  observation. 

The  oil-drops  observed  in  the  chromatophores  of  older 


SPECIAL   METHODS, 


209 


leaves  especially  are  usually  pretty  deep  yellow.  This  is 
due  to  a  pigment  belonging  to  the  group  of  lipochromes, 
which  is  perhaps  identical  with  xanthin  (cf.  Zimmerman n 
III    102). 

4.  The   Eye-spot. 

365.  The  name  eye-spot  or  stigma  is  given  to  the  red  or 
brownish  body  observed  in  various  motile  Alga;  and  swarm- 
spores,  which  usually  occur  singly  at  the  forward  end  of  the 
organism.  These  bodies  correspond  with  the  chromato- 
phores  in  possessing  a  protoplasmic  stroma  filled  with  pig- 
ment. This  pigment  appears,  according  to  the  investiga- 
tions which  have  been  made,  to  be  identical  with  carotin 
(§  170)  in  the  green  algae  which  have  an  eye-spot  ;  at  least,, 
it  shows,  according  to  Klebs  (I,  30),  the  characteristic  blue 
color  with  sulphuric  acid,  in  Euglena, 

The  study  of  the  eye-spot  has  heretofore  been  carried  on 
almost  exclusively  on  living  material. 

5.  The  Elaioplasts  and  Oil-bodies. 

366.  Wakker  gives  (I,  475)  the  name  elaioplasts  or  oil- 
formers  to  bodies  contained  in  the 
protoplasts,  which  he  has  observed 
in  the  epidermis  of  young  leaves 
and  in  the  superficial  parts  of  young 
stems  and  roots  of  Vanilla plani folia 
(cf.  Fig.  45,  e).  They  consist  of  a 
protoplasmic  stroma  and  imbedded 
oil.  The  latter  is  deeply  stained  by 
alcannin  (§  104)  or  cyanin  (§  no), 
and  escapes  in  drops  from  the  elaio- 
plasts when  they  are  treated  with  an 
aqueous  solution  of  picric  acid, 
with  glacial  acetic  acid,  or  with  con- 
centrated sulphuric  acid  ;  it  is  solu- 
ble in  alcohol  and  in  caustic  potash  solution. 

I  have  lately  recognized  elaioplasts  in  various  other  plant- 
tissues,  especially  in  the  epidermis  of  different  parts  of  the 


Fig.  45. — Epidermal  cell  of  a  very- 
young  leaf  of  Vanilla  plani/olia. 
e,  elaioplast  ;  /,  leucoplasts  ;  2, 
nucleus.     After  Wakker. 


2IO 


BOTANICAL   MICROTECHNIQUE. 


■flower  of  Funkia,  Ornithogalum,  Agave,  Draccena,  and  others 
ici.  Zimmermann  VI,  185). 

367.  For  fixing  the  elaioplasts  Wakker  recommends  es- 
pecially a  concentrated  aqueous  solution  of  picric  acid.  He 
obtained  a  beautiful  double  staining  of  sections  fixed  with 
this  fluid  by  using  aniline  blue  and  alcannin  in  the  fol- 
lowing way.  He  added  alcanna  tincture  in  drops  to  a  dark 
blue  solution  of  aniline  blue  in  water,  until  the  fluid  had 
taken  a  dark  purple-red  color,  like  that  of  haematoxylin. 
He  left  the  sections  20  hours  in  this  mixture  and  then  ex- 
amined them  in  glycerine.  The  protoplasm  was  then  light 
blue,  the  nucleus  and  chromatophores  dark  blue,  the  oil  a 
fine  red,  and  the  elaioplast  dark  purple.  These  colors  keep 
for  a  long  time  in  neutral  glycerine-gelatine. 

368.  Wakker  (I,  482)  includes  among  the  elaioplasts  also 
the  oil-bodies  of  the  Hepaticce,  first  described  by  Pfeffer 
(VI).  He  was  able  to  show  by  abnormal  plasmolysis  with 
a  20j^  solution  of  saltpeter  (cf.  §§  432  and  436)  that  these 
always  lie  in  the  protoplasm.  To  render  the  protoplasmic 
wall  of  the  oil-bodies  visible,  Wakker  recommends,  besides 
the  dilute  alcohol  used  for  the  purpose  by  Pfeffer,  a  concen- 
trated aqueous  solution  of  picric  acid.  In  this  case  the 
access  of  the  acid  must  be  followed  with  the  microscope, 

since  a  flattening  of  the  protoplas- 
mic wall  of  the  oil-body  usually  re- 
sults from  its  long  action. 

369.  The  fat-bodies  and  oil-bodies 
described  by  Radlkofer  (I  and  II), 
Monteverde  (I),  and  Solereder  (I) 
are  perhaps  related  to  the  oil-bodies 
of  the  Liverworts.  They  have  been 
observed  by  these  authors  in  the 
palisade  and  spongy  parenchyma  of 
various  members  of  the  Cordiacece, 
CombretacecB,  Cinchonece^  Sapotacece^ 
SapindacecB,  Graminece,  Gaertneracec^y 
and  Rtibiacece.  They  usually  occur 
singly  in  each  cell,  and  have  always  about  the  same  size  in 


Fig.  46.  —  Mesophyll-cell  from 
the  leaf  of  A  vena  orientalis.  c, 
chloroplasts;  z,  nucleus;  o, 
oil-drops. 


SPECIAL   METHODS.  211 

the  same  individual.  In  the  mesophyli  of  Avena  orientalis 
they  are  of  about  the  same  size  as  the  chloroplasts  (cf.  Fig. 
46,  0). 

According  to  Monteverde,  they  lie  in  the  protoplasm  and 
are  mostly  isotropic,  but  in  many  plants  are  also  doubly  re- 
fractive, especially  in  dried  tissues.  In  the  grasses  this 
double  refraction  disappears,  according  to  Monteverde,  when 
sections  are  warmed  to  50°-55°  C.  in  water,  but  reappears 
after  a  few  minutes. 

These  oil-bodies  generally  behave  with  reagents  like  true 
fats.  I  have  found  them,  especially  in  Oplisinemis  iinbecillus 
a.nd  £/j/7/ius  ^^tg'auUus,  inso]uh\Q  in  cold  or  hot  water  or  al- 
cohol, but  soluble  in  ether,  petroleum-ether,  chloroform, 
xylol,  and  clove-oil.  They  are  colored  brown  or  black  by 
osmic  acid.  They  are  deeply  stained  by  cyanin  (§  1 10)  and 
alcannin  (§  109).  On  the  other  hand,  they  remain  completely 
unchanged  in  form  after  24  hours  in  a  mixture  of  one  vol- 
ume of  concentrated  caustic  potash  solution  and  one  volume 
of  ammonia  solution,  and  seem  not  to  be  capable  of  saponifi- 
cation in  this  way  (cf.  §  112). 

370.  Besides  these,  Monteverde  found  in  grasses  contain- 
ing crystals  and  in  those  free  from  them,  drops  of  an  oil-like 
appearance,  but  of  wholly  unknown  composition.  They  in- 
crease in  size  in  water,  glycerine,  and  dilute  acids  only  by 
the  formation  of  vacuoles,  but  gradually  dissolve  in  alcohol. 
After  a  long  stay  in  water,  they  become  insoluble  also  in  al- 
cohol. They  dissolve  with  swelling  in  strong  mineral  acids  and 
acetic  acid,  and  instantly  in  caustic  potash,  ammonia,  ether, 
chloroform,  and  chloral  hydrate.  They  are  not  stained  by 
alcanna  tincture,  but  easily  so  by  aniline  dyes.  They  be- 
come brown  with  iodine.  Monteverde  considers  it  probable 
that  they  consist  chiefly  of  resin. 

I  will  mention  here  the  structures  observed  by  Lundstrom 
(I)  in  the  epidermal  cells  of  various  Potamogeton  species, 
which  he  regards  as  oil-drops,  although,  according  to  his 
statements,  they  are  soluble  in  very  dilute  alcohol. 


212 


BO  TANICAL  MICRO  TECHNIQ  UE. 


6.  The  Iridescent  Plates  of  various  Marine  Algae. 
371.  As  was  first  described  in  detail  by  Berthold  (II,  685), 
there  occur  in  the  superficial  cells  of  some  marine  algae  char- 
acteristic iridescent  protoplasmic  plates,  which  are  very  prob- 
ably to  be  considered  as  organs  for  protection  from  toa 
strong  illumination.  In  the  Chylocladice  they  consist  of  a 
strongly  refractive  mass  in  which  small  granules  of  somewhat 
varying  size  are  imbedded  (cf.  Fig.  47,  a,  p).  In  profile  view 
.•  they  show  a  striation  parallel  to  the 

surface  of  the  plates  and  suggest- 
ing their  composition  of  separate 
lamellae  (cf.  Fig.  47,  b).  These 
plates  are  always  very  sharply  dif- 
ferentiated from  the  cytoplasm. 

In  distilled  water  vacuoles  occur 
in  them,  most  probably  in  conse- 
quence of  the  swelling  of  the  glob- 
ules imbedded  in  its  mass;  and 
finally,  the  plates  show  a  completel}r 


■^1 

Fig.  |7.  —  Superficial  cell   of    the    soongy  StruCturC. 
thallus  of  Chylocladta  reflexa,  n,       ^        c>J 


in  surface  view;  S,  in  profile  view. 
/,  iridescent  plate  ;  c,  chromato- 
phores  (X  250).  After  Berthold. 


372.  ¥  or  fixiftg  the  protoplasmic 
plates  Berthold  recommends  chiefly 
a  concentrated  solution  of  iodine  in  sea-water  (cf.  §  301)  or 
osmic  acid  (§  308).  Neither  of  these,  however,  completely 
preserves  the  structure. 

Whether  these  bodies  are  to  be  included  with  elaioplasts,. 
as  has  lately  been  held  to  be  probable  by  Wakker  (I,  488), 
must  be  determined  by  further  researches. 


7.  Microsomes  and  Granula. 

373-  Under  the  term  microsomes  are  usually  included  all 
those  small  and  mostly  globular  bodies  which  are  distin- 
guishable by  their  different  refractive  power  from  the  main 
mass  of  the  cytoplasm.  But  it  can  no  longer  be  doubted 
that  these  include  bodies  of  very  different  composition,  and 
it  is  not  possible  to  speak  of  special  reactions  of  the  micro- 
somes. 


SPECIAL   METHODS.  2 1 5, 

On  the  other  hand,  it  has  been  shown  by  Altmann  (I)  that 
bodies  of  definite  reactions  may  be  quite  generally  recog- 
nized in  the  cytoplasm  of  animal  cells,  which  this  author 
terms  granula  and  regards  as  the  elementary  organisms  of 
the  cell.  Altmann  used,  for  the  demonstration  of  these 
granula,  chiefly  a  fixing  mixture  of  osmic  acid  and  potassiunrt 
bichromate  and  the  acid  fuchsin  staining  method  A,  de^ 
scribed  in  §  345. 

374.  How  far  the  cytoplasm  of  plant-cells  possesses  a  sim- 
ilar granula-structure  cannot  at  present  be  said.  The  writ- 
er's investigations  on  this  point  have  not  yet  reached  any^ 
conclusive  results.  But,  by  the  aid  of  Altmann's  methods,, 
it  can  be  shown  that  certain  bodies  are  widely  distributed  in 
the  cytoplasm  of  the  cells  of  the  assimilating  tissue,  which 
correspond  in  many  respects  with  Altmann's  granula  and 
have  been  termed  at  first  simply  granula  (cf.  Zimmermann,. 

n,  38)- 

These  are  always  colorless  and  are  mostly  little  spheres^ 
which  have,  at  most,  about  the  size  of  the  nucleoli,  in  adult 
cells  (cf.  Fig.  48,  g).  Their  chemical  relations  indicate  that 
they  consist  of  protein-like  substances. 

375.  Y ox  fixing  the  granula  a  concentrated  alcoholic  solu-^ 
tion  of  corrosive  sublimate 
or  of  picric  acid  may  be 
used  (cf.  §§  310  and  303). 
Very  good  results  have 
been  obtained  also  with 
dilute  nitric  acid,  and  I 
used  in  this  case  a  solution 

COntainine:,    in    O?    volumes     Fig.  48— a  cell  from  the  lowest  mesophyll  layer 

^  ■^'  of  Trade sca.ntia-  albijlora,   from   a   prepara- 

of  water,  three  volumes    of        ^^'on   ^^^^   wUh   alcohoUc     picric    acid    and 

stained  by  Altmann's  acid  fuchsin  method,  c. 
chemically  pure   nitric    acid        chloroplasts;   k,  nucleus;  g,  granula. 

of  specific  gravity  1.3,  which  therefore  contained  about  1.5^ 
of  HNO5.  I  allowed  this  solution  to  act  for  24  hours,  and 
then  washed  the  objects  in  running  water  for  24  hours. 

¥  or  s  taming  the  granula  I  formerly  used  almost  exclusively 
Altmann's  acid  fuchsin  method  (§  345),  but  have  lately  con^ 
vinced  myself  that  the  other  acid  fuchsin  methods  (§§  346 


:2I4  BOTANICAL  MICROTECHNIQUE. 


incr    ■ 


^nd  347)  give  very  good  results.  Especially  after  fixing 
-with  nitric  acid,  preparations  may  be  pretty  easily  obtained 
in  which  the  granula  are  still  deeply  colored,  while  the  much 
4arger  chromatophores  are  wholly  decolorized.  I  can  recom- 
jnend  the  leaves  of  Tradescantia  albiflora  as  suitable  objects 
for  study,  as  they  contain  comparatively  large  granula, 
especially  in  the  spongy  parenchyma  (cf.  Fig.  48,  g), 

[Crato  (I)  has  lately  observed,  in  Chcetopteris  and  other 
plants,  certain  structures,  hitherto  included  under  the  gen- 
-eral  term  microsomes,  which  he  regards  as  special  organs 
of  the  cell  and  calls  physodes.  For  further  details,  his 
-account  of  them  may  be  consulted.] 

8.  The  Cilia. 

376.  The  cilia,  which  occur  on  most  of  the  freely  motile 
lower  organisms  and  are  always  directly  connected  with  the 
protoplasm,  are  often  so  fine  that  during  their  active  motion 
they  can  be  recognized  with  difficulty  or  not  at  all,  even  with 
the  best  objectives. 

377.  In  many  cases  the  cilia  may  be  made  better  visible 
by  bringing  the  organisms  to  rest  by  quickly  killing  them. 
For  this  purpose,  the  vapor  of  osniic  acid  or  i^  osmic  acid, 
\<^  chromic  acid,  or  the  solution  of  iodine  and  potassium 
iodide  may  be  used.  The  cilia  often  appear  sharpl}^  also, 
if  a  drop  containing  the  organisms  be  allowed  to  dry  upon 
the  slide. 

378.  If  the  position  of  the  cilia  is  to  be  determined  while 
jn  motion,  fine  granules  of  carmine,  or  the  like,  may  be 
-added  to  the  fluid  containing  the  organisms,  according  to 
the  method  proposed  by  Butschli  (I,  7).  The  movements 
of  these  granules  will  show  the  cilia-bearing  end. 

379.  Recently  staining  methods  have  also  been  used  for 
the  recognition  of  cilia. 

Migula  (I,  76)  obtained  a  fine  staining  of  the  cilia  of 
4^onium  pectorale  by  using  the  following  method :  A  very 
rsmall  drop  of  a  concentrated  alcoholic  solution  of  cyanin 
was  added  to  the  living  specimens,  and,  after  a  time,  enough 


SPECIAL   METHODS.  2\% 

water  was  added  to  precipitate  the  cyanin  not  taken  up  by 
the  organisms,  in  granular  form.  The  cilia,  as  well  as  the 
rest  of  the  protoplasm,  are  colored  at  first  pale  blue,  but 
after  the  addition  of  water,  deep  violet.  These  methods 
have  given  me  no  favorable  results,  when  used  on  various 
algae. 

380.  But  I  have  obtained  a  very  deep  staining  of  the  cilia 
in  ChlamydomonaSy  Pandorina^  and  Chromophytum  by  the 
following  method,  which  is  essentially  similar  to  methods, 
used  for  staining  the  cilia  of  Bacteria  (cf.  §  476).  The 
objects  were  first  fixed  in  the  hanging  drop  on  the  slide 
by  the  fumes  of  osmic  acid  (cf.  §  308),  and  then  allowed  to- 
dry  ;  then  a  drop  of  a  20^  aqueous  tannin  solution  is  added, 
and  washed  off  with  water  in  five  minutes  or  later.  The 
slide  is  then  plunged  in  a  concentrated  aqueous  solution  of 
fuchsin,*  in  which  it  remains  a  quarter  of  an  hour  or  longer. 
The  fuchsin  solution  is  now  washed  off  with  water,  the: 
preparation  is  again  allowed  to  dry,  and  finally  a  drop  of 
balsam  and  a  cover-glass  are  placed  upon  it.  I  have  ob- 
tained in  this  way  very  beautiful  permanent  preparations  \\x 
which  the  cilia  were  stained  bright  red. 

[I  have  obtained  very  satisfactory  stainings  of  the  cilia  of 
the  zoospores  of  various  algae  and  fungi  by  adding  to  the 
water  containing  them  a  drop  or  two  of  a  i^  solution  of 
osmic  acid,  and  then  the  same  amount  of  a  strong  solution 
in  alcohol  of  equal  parts  fuchsin  and  methyl  violet.  This 
stains  the  cilia  deep  red  almost  at  once ;  and  the  osmic  acid- 
need  not  be  removed  before  adding  the  stain.] 

9.  Protein  Grains. 

381.  The  investigation  of  the  aleurone  or  protein  grains 
which  occur  in  the  seeds  of  all  the  higher  plants  is  best  con- 
ducted, in  oily  seeds,  after  the  removal  of  the  oil,  which  is 
often  a  great  hindrance  to  their  study.     This  may  be  ac- 

*  Carbol-fuchsin  (§  468)  is  especially  useful,  as  it  stains  deeply  in  a  few 
minutes. 


2l6  BOTANICAL   MICROTECHNIQUE. 

complished  in  many  cases,  as  in  Ricifius,  by  placing  the 
■sections  to  be  studied,  for  five  minutes  or  longer,  in  absolute 
alcohol.  Less  soluble  fats  may  be  extracted  with  ether  or 
with  a  mixture  of  equal  parts  ether  and  alcohol.  Further 
methods  of  study  will  depend  upon  the  constituent  of  the 
protein  grain  which  it  is  chiefly  desired  to  make  visible  ; 
for  there  may  be  distinguished  in  them  a  proteid  funda- 
mental mass  and  various  inclusions,  protein  crystalloids, 
globoids,  and  crystals  of  calcium  oxalate.  These  separate 
constituents,  which  are  not  found  simultaneously  in  all 
protein  grains,  but  yet  are  widely  distributed,  will  be  dis- 
cussed in  order. 

[But  it  may  be  well  to  give  first  two  general  methods 
recommended  by  Krasser  (III)  for  making  permanent  prep- 
-arations  to  show  the  grains  and  their  inclusions  in  general. 

1.  The  sections  are  fixed  with  an  alcoholic  solution  of 
picric  acid,  rinsed  with  alcohol,  stained  in  an  alcoholic 
€Osin  solution,  ** toned"  with  alcohol,  cleared  with  clove-oil, 
and  mounted  in  balsam.  The  fundamental  mass  is  stained 
•dark  red,  the  crystalloid  is  yellow,  and  the  globoid  is  colorless 
•or  reddish. 

2.  The  sections  are  simultaneously  fixed  and  stained  in  a 
•concentrated  solution  of  nigrosin  in  an  alcoholic  picric-acid 
solution.  When  the  fundamental  substance  appears  blue, 
which  may  be  determined  by  examining  a  section  in  abso- 
lute alcohol  with  the  microscope,  the  sections  are  washed  in 
alcohol,  cleared  quickly  in  clove-oil,  and  mounted  in  balsam. 
This  treatment  stains  the  fundamental  mass  blue,  the 
crystalloid  yellowish  green,  and  leaves  the  globoid  color- 
less.] 

a.  The  Fundamental  Mass. 

382.  The  fundamental  mass  of  the  protein  grains,  which 
sometimes  forms  the  bulk  of  the  whole  grain  and  some- 
times only  a  thin  envelope  about  the  various  inclusions, 
consists  of  proteid  substances  and  is  well  suited  for  the 
study  of  the  reactions  of  protein,  detailed  in  §§  224  to  234. 


SPECIAL    METHODS.  21/ 

It  is  also  always  readily  soluble  in  dilute  caustic  potash  or 
ammonia  solution  and  in  sodium  phosphate.  The  last- 
named  reagent  in  concentrated  aqueous  solution  is  especially 
recommended  by  Liidtke  (I,  73). 

The  fundamental  mass  of  the  protein  granules  behaves 
very  differently  in  different  plants.  In  many,  as  m  Pceonia, 
it  is  soluble  in  water  ;  in  others  it  is  insoluble  in  it.  It  shows 
similar  relations  with  a  lofc  solution  of  common  salt  and  a 
i^  sodium  carbonate  solution,  differing  with  the  species  of 
plant.  The  protein  grains  which  are  soluble  in  water  are 
best  examined  in  alcohol  or  glycerine  ;  and,  by  the  gradual 
addition  of  water,  their  solution  may  be  observed  under  the 
microscope. 

383.  But  the  protein  grains  may  be  made  insoluble  by 
fixing  media,  for  instance  by  an  alcoholic  solution  of  corro- 
sive sublimate  or  of  picric  acid.  Objects  fixed  in  the  latter 
fluid  may  be  directly  preserved  in  balsam.  But  it  is  also 
■easy  to  stain  the  protein  grains  after  washing  out  the  fixing 
fluid,  and  for  this  purpose  an  aqueous  solution  of  eosin  is 
very  useful. 

384.  The  fundamental  mass  of  the  protein  grains  is 
bounded  externally,  as  well  as  against  the  inclusions,  by  a 
delicate  pellicle  which  is  distinguished  from  the  remaining 
substance  of  the  protein  grain  by  its  insolubility  in  dilute 
alkalies  and  acids,  but,  as  has  been  shown  by  Pfeffer  (I,  449), 
also  consists  of  albuminoid  materials.  According  to  Pfeffer, 
it  may  be  well  observed  by  gradually  dissolving  the  funda- 
mental mass  or  the  inclusions  by  the  addition  of  very  dilute 
caustic  potash,  acetic  acid,  or  hydrochloric  acid.  Ludtke 
has  lately  recommended  lime-water  for  the  same  purpose, 
as  it  first  dissolves  the  fundamental  mass  of  the  grain,  while 
the  membrane  becomes  sharply  visible  and  then  dissolves 
after  a  preliminary  swelling. 

b.  The  Protein  Crystalloids. 

385.  The  crystalloids  observed  in  the  protein  grains  of 
many  seeds  consist  always,  like  the  fundamental  mass,  of 


2l8  BOTANICAL   MICROTECHNIQUE. 

proteids,  as  may  easily  be  shown  by  the  aid  of  the  reactions 
described  in  §§  224  to  234.  When  examined  in  alcohol  or 
glycerine,  they  are  usually  hardly  or  not  at  all  distinguish- 
able from  the  fundamental  mass,  since  they  have  about  the 
same  refractive  index  as  it  (Fig.  49,  II,  a).  But  after  beings 
placed  in  water,  in  which  the  crystalloids  are  always  insoluble, 

they  show  clearly  in  consequence 
of  their  greater  density  (Fig.  49,. 
II,  b).  To  distinguish  them  from 
the  globoids  and  calcium  oxalate 
crystals,  one  may  make  use  of  their 
ready  solubility  in  very  dilute  caus- 
tic potash  and  their  power  of  be- 
coming yellow  or  brown  in  a  solu- 
tion of  iodine  and  potassium  iodide,, 
according  to  its  strength. 
^'onS'^^WoTt^J^^^^  Ludtke  (I,  J'j)  has  lately  recom- 

layerr^^Dl^awn  froS  picric  add    mended    a    Concentrated    aqueous 

I I^Proteln  grains  from  the  endo-  Solution     of     Soduini    pJlOSpkate      for 

sperm     of     Ricinus    communis.,  .                            .    .              /-              ^     11     •  i 

«rin  alcohol,  b,  on  the  addition  the  rCCOgUltlOn  Of  Crystalloids,  SlUCC 

of  iodine-potassium-iodide  solu-  ^.                        .          1     1  1        •        -^           1   -i           n 

tion  after  treatment  with  aico-    they  are   insoluble   m   it,  while   all 

hoi.     A,  crystalloid;  ^,  globoid.  .  ^-.^  ^  e     ^\  .     - 

other  constituents  of  the  protein 
grain  are  dissolved  by  it,  though  sometimes  only  after 
several  hours. 

The  crystalloids  with  distinct  faces  belong,  according  to 
Schimper  (I),  partly  to  the  isometric  and  partly  to  the  hex- 
agonal crystal-system  (cf.  Figs.  49,  II,  <^  and  50,  II  and  III) ; 
and  those  of  the  latter  system  have  a  feeble  doubly  refractive 
power. 

386.  Eosin  is  very  well  adapted  for  staining  the  fixed 
crystalloids.  Acid  fuchsin  may  also  be  used  for  the  same 
purpose  according  to  one  of  the  methods  given  in  §§  345- 
347.  These  dyes  give  a  very  pure  and  deep  staining  of  the 
crystalloids,  especially  after  fixing  with  corrosive  sublimate. 

387.  Recently  Overton  (II,  5)  and  Poulsen  (II)  have  given 
methods  for  staining  crystalloids.  Overton  places  sections 
of  the  endosperm  of  Ricinus,  hardened  with  alcohol,  first  ia 


SPECIAL   METHODS,  219 

a  dilute  aqueous  solution  of  tannin  for  ten  minutes,  and 
then,  after  careful  washing,  in  2^  osmic  acid.  The  crystal- 
loids are  stained  a  beautiful  brown  by  these  reagents.  After 
washing  out  the  osmic  acid,  the  preparations  may  be  pre- 
served in  glycerine. 

Poulsen  (II,  548)  places  the  sections  first  In  alcohol  for 
24  hours,  then  for  an  hour  in  a  25^  aqueous  solution  of 
tannin,  and  finally,  after  washing  this  out  with  water,  in  an 
aqueous  solution  of  potassium  bichromate,  in  which  he 
leaves  them  until  they  are  brown  or  yellowish.  For  the 
preservation  of  these  preparations,  in  which  the  aleurone 
grains  should  be  quite  transparent,  Poulsen  recommends 
glycerine. 

According  to  another  method  also  recommended  by 
Poulsen,  the  sections,  treated  in  the  same  way  with  alcohol 
and  tannin,  are  placed,  after  washing,  for  an  hour  in  a  lO- 
2oio  aqueous  solution  of  ferrous  sulphate.  The  preparations 
are  then  washed  and  transferred  to  balsam  in  the  usual  way. 
The  crystalloids  then  appear  deep  blue,  almost  black. 


c.  The  Globoids. 

388.  The  globoids  consist,  according  to  Pfeffer's  researches 
(I,  472),  of  the  calcium  and  magnesium  salt  of  an  organically 
combined  phosphoric  acid.  They  do  not  occur  in  all  protein 
grains  but,  according  to  Pfeffer's  investigations,  are  not 
wholly  absent  from  any  seed.  They  are  sometimes  more 
or  less  precisely  globular  in  form,  as  in  Pceonia  and  Ricinus 
(cf.  Fig.  49,  I  and  11,^),  sometimes  irregular,  biscuit-shaped, 
or  clustered,  as  in  Bertholletia  excelsa  (Fig.  50,  I).  The  rela- 
tive and  absolute  size  may  vary  very  greatly  in  the  same 
seed.  For  example,  the  protein  grains  in  the  innermost 
layers  of  the  endosperm  of  Pmonia  are  quite  free  of  glo- 
boids, while  their  size  increases  regularly  toward  the  outside 
(cf.  Fig.  49,  I,  a-c). 

389.  In    oil    or   Canada   balsam    the   globoids   have    the 


220  BOTANICAL   MICROTECHNIQUE. 


\3 


appearance  of  vacuoles  (Fig.  49,  II,  a\  because  they  have  a 

lower  refractive   index  than   these 

/                    "K  media.      They    may    be    best    ob- 

^     d      dT^^  served    by   dissolving    the    funda- 

C^  ©     cjr      W       J  mental   mass   of   the   protein-grain 

^^j^  and  the  crystalloids  contained  in  it 

Jf                  jfT  with  dilute,  about  i^,  caustic  potash 

0        ^^  /^  solution,  from    sections    previously 

r^        ^^  ^^  deprived  of  their  fat  by  alcohol  or 

•^  ether-alcohol.     There  remain  then 

"""oidl?-:^?' §lwS^/^^^  in  the  space  formerly  occupied  by 

III,  proteln-Krain  of  Eleeis  gut-       .  ...  .  1.1  1     1       •  1 

neensis.  ivf  protein  grains  of  the  protcm-gram  Only  the  globoids 

Vitis  vini/era :  g,  gloBoid  with  ,  ,    .  ,     ,  ^    , 

calcium  oxalate  crystal  in  the  and  any  calcmm  oxalatc  crystals 
that  may  be  present.  To  distin- 
guish between  these  two  constituents,  polarized  light  may 
be  used.  The  globoids  are  amorphous  and  therefore  iso- 
tropic, while  the  oxalate  crystals  (cf.  §  392)  are  strongly 
■doubly  refractive. 

For  the  same  purpose,  a  dilute,  about  i^,  acetic  acid 
may  be  used,  in  which  the  crystals  are  insoluble,  while  the 
globoids  are  quickly  dissolved  by  it.  In  concentrated  acetic 
acid  the  globoids  are  soluble  with  much  greater  difficulty. 

In  a  concentrated  aqueous  solution  of  sodium  pJiosphaie 
the  globoids  are  completely  soluble,  according  to  Liidtke 
(I,  79),  even  after  treatment  with  corrosive  sublimate.  But 
this  solution  requires  several  hours,  and  the  larger  globoids, 
like  those  from  the  seed  of  Vitis  vinifera,  show  during  solu- 
tion an  evident  stratification  which  gradually  penetrates  from 
without  inwards.  Ludtke  also  observed  similar  stratifica- 
tions when  he  allowed  dilute  caustic  potash  or  lime-water  to 
act  for  a  long  time  on  the  globoids. 

It  may  be  remarked  here  that  the  globoids  are  also  dis- 
solved by  picric  acid.  The  protein-grains,  however,  preserve 
their  original  form  completely  in  this  fluid,  and  cavities  may 
be  seen  in  them  which  have  exactly  the  forms  of  the  dis- 
solved globoids. 

390.  Pfeffer  (I,  472)  used  the  following  reactions  for  the 
recognition  of  the  chemical  composition  of  the  globoids. 


SPECIAL   METHODS.  221 

The  presence  of  organic  substance  in  them  is  shown  by  the 
fact  that  isolated  globoids  blacken  strongly  on  heating. 
They  may  be  easily  obtained  by  moving  about  on  the  cover- 
glass  sections  which  have  been  freed  from  fats  and  proteids 
by  successive  treatment  with  ether-alcohol,  i^  caustic  pot- 
ash, and  water.  To  obtain  a  pure  white  ash  from  the 
globoids,  very  strong  heating  is  necessary. 

If  the  residue  left  after  strong  heating  be  treated  with  an 
ammoniacal  solution  of  ammonium  chloride,  the  character- 
istic crystals  of  ammonio-magnesium  phosphate  are  formed. 
This  shows  at  once  the  presence  of  phosphoric  acid  and 
magnesium  in  the  globoids. 

But  if  the  globoids  be  treated  with  the  ammoniacal  am- 
monium chloride  solution  before  being  heated,  no  formation 
of  ammonio-magnesium  phosphate  crystals  occurs,  evidently 
because  the  organically  combined  phosphoric  acid  behaves 
differently  from  the  phosphoric  acid  set  free  by  heating. 
But  the  formation  of  large  quantities  of  crystals  of  the  double 
salt  mentioned  takes  place  when  sections,  freed  as  above 
from  fats  and  proteids,  are  treated  with  a  mixture  of  an 
ammoniacal  solution  of  ammonium  chloride  and  sodium 
phosphate.  I  have  used  in  this  case,  with  good  results,  a 
reagent  containing  lo  parts  of  the  two  salts  named  and  lO 
parts  of  the  officinal  ammonia  solution  *  to  lOO  parts  of 
water. 

391.  The  presence  of  calcium  was  shown  by  Pfeffer  (I, 
473)  by  the  addition  of  an  ammoniacal  solution  of  ammonium 
chloride  and  ammonium  oxalate  to  the  unchanged  globoids. 
There  are  then  gradually  formed  the  characteristic  crystals 
of  calcium  oxalate.  By  the  addition  of  sulphuric  acid, 
the  formation  of  the  characteristic  gypsum  needles  can  be 
brought  about. 

d.  Crystals, 

392.  The  crystals  observed  within  the  protein-grains  con- 
sists, like  nearly  all  crystals  observed  within  the  vegetable 

*  [This  is  of  16°  Baum6,  spec,  gravity  .960.] 


222 


BOTA  NIC  A  L   MICE  O  TK  CI/N  I Q  UE. 


organism,  of  calcium  oxalate.  For  their  reactions  §§  85  \.<y 
88  may  be  consulted.  It  may  be  observed  here  that  calcium 
oxalate  crystals  can  occur  even  in  the  interiors  of  the  glo- 
boids, as,  for  example,  inside  of  the  large  protein-grains  of 
the  seed  of  Vitis  viiiifera  (cf.  Fig.  50,  IV,  ^). 

10.  Protein  Crystalloids. 

393.  The  protein  crystalloids  contained  in  the  cytoplasm 
or  in  the  cell-sap  agree  essentially  with  the  crystalloids 
contained  within  the  nucleus,  the  chromatophores,  and  the 
protein-grains  (cf.  §§  343,  359,  and  385).  Besides  very 
regular  forms,  like  those  from  the  tubers  of  Solatium  tiibe^ 
rosiim  (Fig.  38,  k)  or  from  the  epidermis  of  the  leaf  of 
Polypoduim  irreoides  (Fig.  51,  I,  k)^  one  finds  also  spindle^ 


Fig,  si.— I,  epidermal  cell  of  the  lower  face  of  the  leaf  of  Polypodium  irreoides;  k,  crys- 
talloid :  z,  nucleus;  c,  chloroplasts.  II,  crystalloids  from  the  wall  of  the  ovary  of 
Gratiola  officinalis.  III.  crystalloid  (k),  chloroplasts  (r),  and  granula  {g)  from  a 
spongy-parenchyma  cell  of  Passifiora  caerulea..  Iv,  crystalloids  from  the  subepidermal 
parenchyma  of  the  leaf  of  Vanda  furva. 

shaped  or  needle-like  forms,  or  such  as  are  variously  bent 
(Fig.  51,  II-IV).  Molisch  (II)  observed  completely  ring- 
shaped  crystalloids  in  the  leaves  of  various  species  of  Epi- 
pJiylluin. 

For  the  recognition  of  these  crystalloids  the  staining 
methods  detailed  in  §§  345  to  347  may  best"  supplement  the 
study  of  living  material. 

394.  As  has  been  recognized  especially  by  J.   Klein  (I), 


SPECIA  L   ME  THOD  S,  22  3 

true  protein  crystalloids  occur  in  many  marine  algae.  These 
must  not  be  confused  with  the  so-called  rhodospermin-crys- 
talloids  which  are  formed  after  the  algae  have  been  killed  in 
a  solution  of  common  salt  or  in  spirit. 

395.  Rhodosperniin  consists  chiefly  of  hexagonal  prisms  or 
plates  which  are  colored  deep  red  (Fig.  52).  They  are 
insoluble  in  water,  alcohol,  glycerine,  sulphuric,  nitric,  hydro- 
chloric, and  acetic  acids,  and  in  alkalies.  They  become 
gradually  destroyed  and  invisible  on  boiling  in  sulphuric 
acid,  hydrochloric  acid,  or  potash.  Iodine  colors  them  at 
first  golden  yellow  and  later  deep  brown-yellow.  They 
are  not  colored  by  concen- 

trated   nitric  acid,  but   on  ^  ^ 

the    subsequent     addition       r — ^  /\ 

of  ammonia  become  most  ^ )\-.^J^ 

clearly     yellow.       Rhodo-  ^  (j  fj 
spermin  often  swells  mark-  ' 

edly  in  a  potash  solution,  Yig.  52.-Rhodospermin  crystals  from  Cera- 
U  4.  ^^  ,4-^^^^-^  ^^^:»,  *-^  J*-^  mium  rubrutn.  rt,  formed  in  the  cortical  tis- 
DUt    contracts    agam    to    its       sue;  ^,  rhodospermin  formed  free  in  the  fluid. 

original  bulk  on  the  addi- 
tion of  acids,  while,  at  the  same  time,  the  color  which  has 
disappeared  in  the  potash  reappears  (cf.  J.  Klein  I,  55). 

Whether  we  are  justified  by  these  reactions  in  including 
rhodospermin  among  the  proteids,  as  is  commonly  done, 
seems  to  me  at  least  doubtful.  But,  at  any  rate,  the  rhodo- 
spermin crystalloids  do  not  belong  in  the  same  category 
with  other  crystalloids,  for  they  represent,  as  has  already 
been  remarked,  an  artificial  product  arising  through  the 
action  of  reagents. 

II.  Rhabdoids  (Plastoids). 

396.  Gardiner  observed  (I)  in  most  of  the  epidermal  cells 
of  Drosera  dichotoma,  as  well  as  in  those  of  Dioncea,  spindle- 
shaped  or  needle-shaped  bodies  which  he  first  termed  plas- 
toids and  later,  rhabdoids.^  These  usually  occur  singly  in 
the  cells,  which  'they  cross  diagonally.     They  are  fixed  by 

*  From  r}  f)dftSo<i,  the  rod. 


224  BOTANICAL   MICROTECHNIQUE. 

alcohol,  chromic  acid,  and  picric  acid.  After  fixing  with  the 
last-named  acid,  they  may  be  deeply  stained  with  Hof- 
mann's  blue  ;  but  in  dilute  alcohol  they  swell  and  finally 
disappear  wholly.  They  are  also  disorganized  by  iodine  and 
take  a  spherical  form. 

After  stimulation  of  the  leaves  the  rhabdoids  contract  and 
become  rounded  or  fall  into  several  pieces,  which  become  at 
first  lens-shaped,  but  later  always  more  spherical. 

After  longer  stimulation  the  rhabdoids  decrease  markedly 
in  size  ;  they  are  therefore  regarded  by  Gardiner  as  reserve 
materials. 

Whether  these  structures  should  be  simply  included  with 
the  crystalloids  cannot  be  determined  from  our  present 
knowledge. 

12.  The  Acanthospheres  of  the  Characeae. 

397.  The  acanthospheres,*  or  ciliate  bodies,  observed  in 
the  cells  of  various  species  of  Nitella,  have  already  received 
various  explanations  (cf.  Zimmermann  I,  73).  According  to 
Overton's  recent  researches  (II),  they  consist  of  albuminoid 
substances  which  most  probably  possess  a  crystalline  struc- 
ture and  are  partly  united  with  tannins. 

398.  For  recognizing  the  presence  of  tannin  in  the  acan- 
thospheres, Overton  used  potassium  bichromate,  osmic  acid, 
and  staining  intra  vitam  with  methylene  blue  (cf.  §§  201,  203, 
and  208).  He  deduced  their  proteid  nature  from  their  be- 
havior with  iodine  and  potassium  iodide  solution,  Raspail's 
reagent,  and  Hartig's  potassium  ferrocyanide-ferric-chloride 
reagent  (cf.  §§  224,  227,  and  230).  He  also  stained  objects 
fixed  with  an  alcoholic  sublimate-solution  with  borax-carmine 
and  an  aqueous  solution  of  fuchsin.  The  crystalline  struc- 
ture of  the  acanthospheres  is  best  shown  by  the  use  of  the 
former  stain  and  mounting  in  balsam  Tolu. 

The  difficult  solubility  of  the  acanthospheres  in  acids  and 

*  [As  I  am  not  aware  that  any  English  name  has  been  applied  to  these 
bodies,  I  propose  this  Greek  equivalent  of  the  German  name,  StacheK 
kUgeln.] 


SPECIAL    METHODS.  22$ 

alkalies  is  especially  remarkable.  These  bodies  remain 
almost  unchanged  in  concentrated  sulphuric,  hydrochloric, 
and  nitric  acids,  and  in  glacial  acetic  acid,  according  to 
Overton  (II,  4).  Cold  caustic  soda  also  fails  to  attack  them, 
but  on  boiling  in  this  solution,  the  spiny  envelope  gradually 
disappears  and  the  interior  assumes  a  spongy  structure. 

399.  It  may  also  be  remarked  that  Overton  has  found  in 
the  cells  of  Nitella  syncarpa,  besides  these  acanthospheres, 
vesicles  as  clear  as  water  which  show  the  same  chemical  re- 
lations as  they  ;  and  intermediate  stages  between  the  two 
bodies  were  to  be  found.  In  the  species  of  Char  a  that  he 
studied,  Overton  found  only  such  spineless  bodies  ;  these 
are  very  probably  identical  with  the  strongly  staining  bodies 
recognized  by  the  writer  in  the  cells  of  a  species  of  Chara 
not  definitely  determined,  after  fixing  with  nitric  acid  and, 
staining  with  acid  fuchsin  (cf.  Zimmermann  II,  51). 

13.  Starch-grains  and  Related  Bodies 

a.  Starch. 

400.  The  chemical  composition  of  starch  corresponds  to 
the  formula  CgHjoO^;  but  it  is  not  yet  determined  whether 
we  have  to  do  with  a  completely  uniform  compound  in  the 
starch-grains  or  not. 

The  form  of  starch-grains  shows  the  greatest  diversity  in 
different  plants.  But  in  a  given  organ  of  the  same  species 
of  plant,  only  slight  variations  are  observed,  except  such 
as  are  due  to  the  different  stages  of  development  of  the 
grains.  Some  of  the  characteristic  forms  of  starch-grains 
are  shown  in  Fig.  53.  Figures  I,  III,  and  V  represent 
simple  grains  from  Carina,  Lathrcea,  and  Euphorbia,  The 
last  two  sorts  are  especially  distinguished  by  their  charac- 
teristic form.  Figure  IV  shows  a  cell  from  the  horny  part 
of  the  endosperm  of  corn,  in  which  the  starch-grains  lie 
almost  in  contact.  Figures  II  and  VI,  finally,  show  the  so- 
called  compound  grains  of  Beta  and  Smilax.  The  former 
consist  of  an  immense  number  of  component  grains. 

401.  The  larger  starch-grains  show  usually  a  more  or  less 


226 


BO  TA  NIC  A  L   MICRO  TECIINIQ  UE, 


distinct  stratification,  which  is  certainly  due  to  varying  water- 
content.  This  may  be  shown  by  exannining  moist  and  dry 
starch-grains  in  Canada  balsam  or  the  like  (cf.  §  297k  and 
Zimmermann  I,  87).  The  silvering  described  in  §  297n  may 
also  be  carried  out  on  starch-grains,  as  Correns  (III,  331) 
has  shown.     It  is  best  done  by  drying  at  50°  C.  starch-grains 


n 


Fig.  53.— I,  starch-grain  from  the  rhizome  of  Canna  indica  (X  300) ;  II,  the  same,  from 
the  seed  of  Beta  vulgaris  (X  500)  ;  III,  the  same,  from  the  scale-leaves  of  Lathrcea 
squamaria  {y^  \<p)  \  IV,  endosperm-cell  of  ZeaMays;  V,  starch-erains  from  the  latex 
of  Euphorbia  spiendens  (X  150);  VI,  the  same,  from  the  Sarsaparilla  root  (X  250). 


obtained  by  scraping  and  cleaned  by  washing  and  decanting. 
When  dry,  the  grains  are  covered  with  a  few  drops  of  a  5JI^ 
solution  of  silver  nitrate,  then  a  large  quanity  of  a  lo^  solu- 
tion of  common  salt  is  added,  and  the  whole  is  exposed  to 
bright  light  until  the  reduction  is  completed.  The  grains 
are  finally  dried  again  on  the  slide  and  mounted  in  Canada 
balsam.  There  are  then  seen  many  granules  of  silver  de- 
posited in  the  more  strongly  swelling  layers  of  most  of  the 
larger  grains. 

402.  In  polarized  light  starch-grains  give  a  characteristic 
figure,  like  that  of  sphaerocrystals ;  but  the  orientation  of 
the  optical  axes  is  opposite  to  that  of  the  sphaerites  of  inulin, 
for  example.  In  excentrically  formed  starch-grains,  the 
middle  of  the  black  cross  is  also  excentric  and  always  corre- 
sponds with  the  organic  centre  of  the  grain. 


SPECIAL   METHODS.  22/ 

403.  Concerning  the  microchemical  relations  of  starch,  it 
may  be  first  remarked  that  it  is  entirely  insoluble  in  cold 
water ;  but  in  hot  water  it  suffers  first  strong  swelling,  and 
on  longer  boiling  it  passes  completely  into  solution  (paste). 
Caustic  potash  also  causes  a  marked  swelling  and  finally 
complete  solution  of  the  starch-grains. 

But  its  becoming  blue  with  iodine  is  especially  character- 
istic of  starch.  This  does  not  take  place  indiscriminately 
with  iodine  solutions,  for  all  solutions  containing  hydriodic 
acid  or  potassium  iodide  give  it  rather  violet-brown  tones. 
A  pure  blue  coloring  of  starch-grains  is  obtained  by  prepar- 
ing an  aqueous  solution  of  iodine  immediately  before  it  is  to 
be  used,  by  adding  a  few  drops  of  an  alcholic  solution,  which 
keeps  indefinitely,  to  a  few  ccm.  of  distilled  water.  The  use 
of  dilute  solutions  is  always  to  be  recommended  for  coloring 
starch-grains,  as  they  often  become  almost  black  in  concen- 
trated solutions.  Swollen  grains,  and  even  paste,  have  the 
same  power  as  unchanged  ones  of  becoming  blue  with  iodine. 
This  color  disappears  on  heating,  but  reappears  on  subse- 
quent cooling,  without  further  addition  of  iodine.  Caustic 
potash  always  at  once  destroys  the  color  by  the  decomposi- 
tion of  the  iodine. 

404.  For  the  recognition  of  very.small  quantities  of  starch 
various  methods  have  been  given,  which  consist  essentially 
in  swelling  the  starch-grains  before  the  addition  of  iodine 
and  destroying  the  rest  of  the  cell-contents.  But  in  most 
cases  it  is  as  well  to  place  thin  sections  in  a  concentrated 
solution  of  iodine  and  potassium  iodide ;  the  deep  black 
starch-grains  then  come  out  sharply  with  high  powers  and 
strong  illuminations. 

405.  In  many  cases,  the  sections  may  well  be  treated 
according  to  the  method  of  A.  Meyer  (II).  After  treatment 
with  a  pretty  dilute  solution  of  iodine  and  potassium  iodide, 
and  the  removal  of  this  reagent,  a  concentrated  aqueous  solu- 
tion of  chloral  hydrate  is  added  to  the  sections,  which 
destroys  the  other  cell-contents  and  causes  the  starch-grains 
to  swell  so  that  they  appear  brighter  and  usually  of  a 
beautiful  blue  color.     But  it  should  be  observed  that  starch 


228  BOTANICAL   MICROTECHNIQUE. 

is  also  finally  decomposed  by  chloral  hydrate,  and  that  verjr 
small  amounts  of  starch,  which  are  naturally  first  attacked,. 
may  be  easily  overlooked,  if  the  sections  are  not  examined 
soon  after  the  addition  of  chlorate  hydrate. 

The  action  of  chloral  hydrate  may  be  hastened  by  heating,, 
but  the  color  of  the  starch,  due  to  iodine,  thereupon  disap- 
pears,  to  reappear  on  cooling  again. 

Eau  de  Javelle  (§  12,4),  recommended  by  Heinricher  (I)  for 
the  recognition  of  starch,  acts  in  the  same  way  as  chloral 
hydrate.  But,  according  to  this  author's  statements,  it 
is  better  to  treat  the  objects  with  it  before  the  addition 
of  iodine,  since  otherwise  the  iodine  hinders  the  destruction 
of  the  protoplasm. 

Both  media,  chloral  hydrate  and  eau  de  Javelle,  may  be 
used  with  advantage  when  it  is  desired  to  demonstrate  the 
distribution  of  starch  in  large  organs,  like  whole  leaves.  It 
is  best,  in  this  case,  to  place  the  objects  in  a  concentrated 
solution  of  chloral  hydrate  containing  iodine,  and  to  heat 
them  to  boiling  in  it.  In  this  way  leaves  at  all  delicate  may 
be  cleared  and  saturated  with  iodine  in  a  few  minutes. 

406.  The  recognition  of  starch  may  be  accomplished  with 
greater  certainty  in  microtome  sections.  For  this  purpose, 
it  is  best  to  use  a  very  concentrated  solution  of  iodine  and 
iodide  of  potassium,  which  stains  the  smallest  starch-grains 
dark  violet  or  quite  black.  These  then  stand  out  sharply,, 
even  in  cells  rich  in  protoplasm,  from  the  brown  proteids.  In 
doubtful  cases,  a  concentrated  chloral  hydrate  solution  may 
be  afterward^  added,  in  which  the  starch-grains  swell  and 
become  of  a  beautiful  blue  color,  while  the  rest  of  the  cell- 
contents  suffer  a  wide-reaching  destruction. 

407.  I  will  remark  here  that  not  all  starch-grains  are 
colored  blue  by  iodine.  But  there  are  some  which  are 
colored  wholly  or  in  part  red  by  iodine,  or  which  take 
intermediate  colors  between  red  and  blue.  It  is  probable,, 
according  to  the  researches  of  Shimoyama  (I)  and  A.  Meyer 
(I),  that  thevarying  behavior  of  these  starch-grains  is  due 
to  the  fact  that  they  contain  greater  or  less  quantities  of 
amylodextrine  and  dextrine  besides  true  starch. 


SPECIAL   METHODS.  229 

Amylodextrine  is  almost  insoluble-  in  cold  water,  but  Is 
easily  dissolved  by  water  at  60°  C,  and  is  not  thrown  down 
from  such  a  solution  on  cooling.  But,  on  evaporation  or 
freezing,  amylodextrine  separates  from  its  solution  in  the 
form  of  characteristic  crystaUine  disks,  the  so-called  "  disco- 
crystals  "  (cf.  W.  Naegeli  I,  and  Naegeli  and  Schwendener 

I,  359). 

408.  Amylodextrine  also  arises  as  an  intermediate  prod- 
uct in  the  transformation  of  starch  into  dextrine  and  malt- 
ose. If  starch-grains  are  heated  for  some  time  at  50°  C.  in 
the  salivary  ferment,  they  lose,  usually  after  a  few  hours,, 
the  power  to  become  blue  with  iodine,  and  take  at  first  a 
violet,  then  a  wine-red,  and  finally  a  pure  yellow  color,  ac- 
cording to  the  length  of  time  the  ferment  has  acted.  Nev- 
ertheless the  grains  which  remain,  the  so-called  "  starch- 
skeletons,"  still  have  the  original  form  of  the  starch-grains 
and  often  show  distinct  stratification.  These  starch-skele- 
tons, consisting  of  amylodextrine,  may  also  be  obtained  by 
the  action  of  dilute  hydrochloric  or  sulphuric  acid  for  years- 
upon  unchanged  starch-grains. 

409.  But  the  solution  of  starch  within  the  living  plant 
takes  place  in  another  way.  As  has  lately  been  described 
in  detail  by  Krabbe  (I),  either  there  occurs  a  regular  solu- 
tion from  without  inwards,  or  local  corrosions  are  formed  in 
the  shape  of  pit-  or  crater-shaped  depressions.  In  small 
grains  pore-canals  are  often  formed,  which  may  penetrate 
to  the  interior  of  the  grain  and  lead  to  the  formation  of  a 
central  cavity. 

Diastase  acts  in  a  similar  manner.  It  may  be  prepared 
by  the  solution  of  malt  extract  in  water.  A  very  useful  fer- 
ment-containing fluid  may  be  prepared  by  adding  to  the 
aqueous  solution  of  commercial  diastase  about  .05^  of  citric 
acid  (cf.  Detmer  I,  197). 

410.  Finally  there  may  be  mentioned  here  a  substance 
commonly  termed  soluble  or  amorphous  starch,  which  agrees 
with  true  starch  in  being  colored  blue  or  violet  to  red  by 
iodine  solutions.  But  it  is  soluble  in  water  and  occurs  only 
in  the  cell-sap  of  the  epidermal  cells  of  a  few  plants ;  for  ex« 


230  BOTANICAL   MICROTECHNIQUE. 

ample,  Saponaria  officinalis.     No  certain  statements  can  be 
made  as  to  its  chemical  composition  (cf.  J.  Dufour  II). 

b.  Floridean  Starch. 

411.  Colorless  granules  have  been  observed  in  the  cells  of 
the  FloridecB,  which  correspond,  as  Van  Tieghem  (I,  804) 
recognized,  in  most  of  their  chemical  characters,  especially 
in  their  behavior  with  caustic  potash  and  hot  water,  with 
ordinary  starch,  and  which  show,  under  the  polarizing  mi- 
croscope, a  similarly  oriented  cross  to  that  of  the  latter. 
With  iodine  these  granules,  which  are  commonly  termed 
Floridean  or  Rhodophycean  starch,  usually  take,  however, 
only  a  yellowish-brown  to  a  brownish-red  color.  According 
to  more  recent  investigations  of  Belzung  (I,  224),  in  many 
FloridccB  the  younger  starch-grains,  especially,  are  colored 
blue  by  iodine.  We  have  at  present  no  more  exact  chem- 
ical studies  of  the  substance  of  Floridean  starch. 

c.  Phseophycean  Starch. 

412.  Schmitz  (I,  154  and  II,  60)  has  observed  in  the  cyto- 
plasm of  the  PhcBophycece  colorless  bodies  quite  insoluble  in 
water,  but  which  are  not  colored  at  all  by  iodine.  Berthold 
has  disputed  (I,  57)  the  occurrence  of  such  bodies. 

d,   Paramylum. 

413.  The  paramylum-grains,  recognized  m  the  cytoplasm 
of  the  Euglence  and  by  Zopf  (I,  17)  in 
the  amoebae  and  cysts  of  Leptophrys 
vorax,  have  mostly  a  disk-shaped  or 
rod-shaped  form  (cf.  Fig.  54,  I) ;  but 
ring-shaped  grains  have  also  been  seen 
(Fig.  54,  II  and  III). 

Klebs  (I,  271)   observed   an  evident 
stratification  in  the  large  paramylum- 

F.o.  54.-Paratnylum.erains.  I.   g»"ains    of    EligiiUa    Ehreubergii,  which 

"fufl^iiZV'uiS'fu/uZ  is,  however,  very  characteristic   in   its 
'/ii:TEhr^enZ'jr.dif:^  arrangement  and  differs  markedly  from 
KiJSs. ''**'''  ^^  '~^*    ^^'"'  that  of  the  starch-grains.     These  para- 
mylum-grains consist,  as  is  shown  in  Fig.  54,  IV,  a-c,  which 


SPECIAL   METHODS.  23 1 

represents  such  a  grain  in  three  different  views,  of  plates 
placed  close  together  and  themselves  composed  of  concen- 
tric rings.  The  whole  grain  therefore  lacks  a  common  cen- 
tre of  stratification.  In  other  paramylum-grains  Klebs  was 
able  to  bring  out  the  stratification  only  by  swelling  media. 

The  paramylum-grains  differ  chemically  from  the  starch- 
grains  in  not  being  colored  by  iodine  solutions  and  in  not 
being  stainable,  in  general.  Klebs  (I,  270)  also  gives  it  as 
characteristic  of  them  that  they  remain  quite  unchanged  in 
5^  caustic  potash  and  do  not  swell,  while  in  even  a  6^  solu- 
tion of  the  same  they  at  once  dissolve  with  strong  swelling. 
We  have  no  more  exact  chemical  analysis  of  these  grains. 

e.  Cellulin-grains. 

414.  The  cellulin-grains  discovered  by  Pringsheim  (I)  in 
the  hyphae  of    various  SaprolegniacecB  have  sometimes  the 
form  of  round  or  polyhedral  plates  and  are  sometimes  more 
globular,  and  often  show  evident  stratification  (cf.  Fig.  5$). 
They  are  not  colored  by  iodine  solutions,  and 
are  even  insoluble  in  concentrated  caustic  pot-     A®S     <^ 
ash  solution,  but  soluble  in  concentrated   sul-      {G\v§)§' 
phuric  acid  and  in  a  solution   of  zmc  chloride.     '^'•~-'  O 
Nothing  is  known  of  their  chemical  constitu-  Fig.  ss.-Ceiiuiin- 

°  grains  of  Lepto- 

tion.  tnitus       lacteus. 

After        Prings- 

Weber  van  Bosse  (I)  discovered  globular  bod-  heim. 
ies  which  agree  completely  with  cellulose  in  their  reactions, 
in  one  of  the  Phyllosiphonacece,  Phytophysa  Treubii.  They 
are  not  colored  by  a  solution  of  iodine  and  iodide  of  potas- 
sium, but  are  colored  blue  by  iodine  and  sulphuric  acid,  and 
violet  by  chloroiodide  of  zinc. 


f.  Fibrosin-bodies. 

415.  Zopf  (III)  found,  in  the  conidia  of  Podosphcera  Oxy- 
acanthcB  and  of  other  Erysiphece,  characteristic  bodies  which 
he  calls  fibrosin-bodies.  They  are  always  imbedded  in  the 
cytoplasm  and  are  sometimes  cup-shaped,  sometimes  in  the 


232  BOTANICAL   MICROTECHNIQUE. 

form  of  a  hollow  cone   or  a  hollow   cylinder  (cf.   Fig.   56). 

Their  largest  diameter  varies  from  2  to 

vv  «      ^F    >^     8  }i:     No  stratification  can  be  observed 

in  them,  even  after  treatment  with  caus- 

J&      @"®   6  ^     ^^^  potash    or    chromic    acid.     For  the 

examination  of  the  fibrosin  bodies  Zopf 

^from^the  cdnfdiTof  °Pojn-  recommcnds   that   they  be  isolated   by 

in  different  views  as  indi    prcssure  on  the  cover-glass,  or  that  the 

cated  by  the  dotted  lines.  ,  ,  ^  ^        -.i 

(xiooo).  After  Zopf.  sporcs  be  made  more  transparent  with 
nitric  acid  or  caustic  potash.  Zopf  has  determined  the 
following  points  as^  to  their  chemical  relations :  They 
swell  in  hot  water  into  roundish  bodies ;  they  are  colored 
neither  by  iodine-potassium-iodide  solution  nor  by  chloro- 
iodide  of  zinc ;  neither  are  they  dissolved  by  the  latter  re- 
agent. In  concentrated  sulphuric  acid  they  are  soluble  with 
•difficulty ;  they  are  not  dissolved  by  nitric  acid,  even  after 
48  hours'  exposure.  In  caustic  potash  they  are  insoluble 
in  the  cold,  but,  on  warming  in  it,  they  swell  up  into  irregu- 
lar, strongly  refractive  bodies.  They  are  insoluble  in  cu- 
prammonia,  alcohol,  ether,  and  chloroform,  are  not  browned 
by  osmic  acid,  and  take  up  no  aniline  dye. 

The  fibrosin-bodies  therefore  agree  most  closely  in  their 
reactions  with  fungus-cellulose,  and  are  distinguished  from 
the  cellulin-grains  especially  by  their  insolubility  in  chloro- 
iodide  of  zinc  and  their  difficult  solubility  in  concentrated 
sulphuric  acid.  A  macrochemical  analysis  has  not  yet  been 
carried  out,  for  obvious  reasons. 


14.  The  Mucus-globules  of  the  Cyanophyceae. 

416.  Several  authors  have  observed  globular  structures  in 
the  cells  of  the  CyanophycecE,  which  always  lie  in  the  periph- 
eral part  of  the  protoplasm  (Fig  57,  s)  and  are  often  es- 
pecially abundant  on  both  sides  of  the  transverse  walls. 
These  may  be  here  termed,  with  Schmitz,  mucus-globules, 
although  no  conclusion  as  to  their  chemical  composition 
is  at  present  possible. 

417.  According  to  the  recent  investigations  of  Zacharias 


SPECIAL   METHODS. 


233 


^III,  p.  12  of  separata),  these  mucus-globules  give  the  follow- 
ing reactions :  They  are  insoluble 
in  alcohol  and  in  ether,  and  are  un- 
changed either  by  boiling  in  distilled 
water  or  by  the  addition  of  a  i^  solu- 
tion of  soda  (NaaCOj).  In  .3^  hydro- 
4:Jiloric  acid  they  swell  up  and  become    Fig.  57.-1,  living  ceii  of  scyto- 

.    .,  1  1    ,1  1.     •  nema\   2,  cell  of  iV<»j/<»c,  after 

invisible,  and  the  same  result    is    pro-        staining  with  acetic  carmine. 

,  ,  ,  .     ^  r  ,  ,  r         •^'     mucus-globules.  After 

duced  by  a  mixture  ot  two  volumes  of  Zacharias. 
concentrated  sulphuric  acid  and  three  volumes  of  water; 
while  they  swell  even  in  a  mixture  of  one  volume  of  sul- 
phuric acid  and  lOO  volumes  of  water,  but  remain  visible. 
3-5^  caustic  potash  solution  causes  swelling  of  the  mucus- 
globules,  but  in  a  i^  solution  they  are  unchanged.  In  a 
solution  of  potassium  ferrocyanide  acidulated  with  acetic 
acid  they  stand  out  sharply  and  take  a  vacuolar  structure. 
In  Millon's  reagent  they  remain  colorless,  and  also  in  iodine- 
glycerine  or  in  chloroiodide  of  zinc.  lodme  and  potassium 
iodide  solution  and  the  above-mentioned  dilute  sulphuric 
acid  cause,  on  the  other  hand,  a  deep  brown  coloring  of  the 
mucus-globules.  Acetic  carmine  colors  them  deep  red,  and 
a  deep  stain  is  produced  by  alum-carmine  and  by  Delafield's 
haematoxylin,  after  treatment  with  alcohol. 

Their  capacity  for  staining  with  haematoxylin  has  been 
disputed  by  Biitschli  (I,  17).  According  to  his  statements, 
£osin  is  especially  useful  for  staining  them.  The  mucus- 
globules  may  be  made  visible,  even  in  haematoxylin  prepa- 
rations, by  subsequent  staining  with  this  agent.  [Hierony- 
mus  (II)  has  studied  bodies  found  in  the  cells  of  Cyanophycece, 
Avhich  he  terms  cyanopJiycin-grains  and  regards  as  identical 
Avith  the  mucus-globules  of  Schmitz.  According  to  his 
statements,  the  bodies  studied  by  him  agree  in  their  reac- 
tions with  those  above  described,  and  he  gives  the  following 
additional  facts  concerning  them  :  They  give  no  proteid 
reaction.  They  are  quickly  dissolved  by  nitric  acid,  by  a 
solution  of  salt,  by  eati  de  Javelle^  chloral  hydrate^  and 
caustic  potash  solution  ;  but  are  insoluble  in  an  artificial 
gastric  juice,  in  carbon  bisulphide,  in  acetic  acid,  and  in  a 


234 


BO  7'ANICAL   MICRO  TECHNIQ  UE. 


cold  solution  of  di-sodium  phosphate  (Na,HPO,);  but  in  a 
boiling  concentrated  solution  of  the  last  they  slowly  dis- 
solve. This  author  states  that  these  bodies  are  formed  of  a 
knot  of  thread,  and  he  regards  them  as  probably  related  ta 
nuclein  (cf.  §  236).] 


15.  Tannin-vesicles. 


418. 


Fig  58.— Cell  of  Meso- 
carfus  sp.  g^  tannin- 
vesicles;/,  pyrenoid; 
2,  nucleus. 


In  the  cells  of  many  ZygneinacecB  are  found  strongly 
refractive  globular  structures,  which,  as 
Pringsheim  (II,  354)  recognized,  contain 
large  quantities  of  tannin  and  are  com- 
monly  termed  tannin-vesicles.  Such  tan- 
nin-vesicles have  also  been  observed  in 
various  Phanerogams  (cf.  af  Klercker  1, 15). 
But,  while  they  are 
usually  present  in 
the  Algae  m  e  n  - 
tioned  in  large 
numbers,  and  are 
of  small  size  (cf. 
Fig-  58,  g\  in  the 
Phanerogams  they 
are  usually  single 
or  only  a  few  in  a 

cell     and     are     of     a  ^"^-  59-— Cell  from  the  base  of 

the  petiole  of  Desmanthus  ple- 

relatively       large       ""^-    ^»  tannin-vesicle;  z,  nu- 


cleus ;  c,  chloroplasts. 


size,  as  in  the  bases 
of  the  petioles  of  Desmanthus  ple7ius  (cf.  P'ig.  59,  g). 

419.  These  tannin-vesicles  always  arise,  as  Klercker  (I^ 
22)  has  .shown,  in  the  protoplasm,  from  which  they  are 
most  probably  separated  by  a  true  precipitation  membrane 
of  albumen  tannate.  Whether  they  contain  other  sub- 
stances than  tannins  cannot  at  present  be  certainly  stated ; 
but,  at  all  events,  they  cannot  contain  dissolved  proteids,  as 
Klercker  (I,  36)  has  shown. 

420.  In  the  investigation   of  the  tannin-vesicles,  all  the 


SPECIAL    METHODS.  235 

tannin  reactions  described  in  §§  199-208  may,  of  course,  be 
used.  Especially  useful  is  Pfeffer's  staining  intra  vitant 
with  methylene  blue  (§  208),  which  may  be  used  with 
particular  advantage  in  developmental  investigations.  As 
suitable  objects  for  study  may  be  suggested  the  cells  of 
Zygnema  and  Mesocarpus  (cf.  Fig.  58),  as  well  as  those  from 
the  root-cap  of  Pistia  Stratiotes  or  from  the  base  of  the 
petiole  of  DcsniantJius  pleniis  (cf.  Fig.  59). 

421.  A  good  fixation  of  this  coloring  may  be  obtained 
by  placing  the  stained  objects  in  a  concentrated  aqueous 
solution  of  picric  acid  for  2-24  hours.  They  are  then 
repeatedly  rinsed  in  pure  water,  then  placed  in  10-20^ 
alcohol,  which  is  gradually  replaced  by  absolute  alcohol  by 
means  of  Schulze's  dehydrating  vessel  (§  16),  then  trans- 
ferred to  a  mixture  of  three  parts  xylol  and  one  part 
alcohol,  finally  to  xylol,  and  then  mounted  in  balsam.*  In 
this  way  I  have  obtained  beautiful  permanent  preparations 
of  Zygneuia,  in  which,  after  six  months,  the  tannin-vesicles 
alone  are  stained  deep  blue  ;  while  threads  preserved  for 
the  same  time  in  glycerine-gelatine  have  almost  entirely  lost 
their  color. 

I  have  not  experimented  as  to  whether  a  solution  of 
ammonium  picrate,  recommended  by  Dogiel  (I)  for  fixing^ 
methylene  blue  stains  in  animal  objects,  is  to  be  preferred 
to  the  watery  solution  of  picric  acid  for  vegetable  prepara- 
tions also. 

To  obtain  permanent  preparations  of  the  tannin-vacuoles, 
af  Klercker  (III)  fixes  the  objects  either  with  Flemming's 
chrom-osmic  acid,  or  with  a  mixture  of  one  volume  of 
Kleinenberg's  picro-sulphuric  acid  and  one  volume  of  a  5^ 
solution  of  potassium  bichromate,  or  with  a  mixture  of 
equal  parts  picro-sulphuric  acid  and  cupric  acetate  solution. 
After  washing,  the  objects  are  imbedded  in  paraffine  in  the 
usual  way.     Finally,  a  much  darker  staining  of  the  tannin 


*  Clove-oil,  phenol,  or  aniline  must  not  be  used  in  the  transfer  to  balsam, 
as  they  at  once  wash  out  the  methylene  blue  stain. 


236  BOTANICAL   MICROTECHNIQUE. 

precipitates  may  be  obtained,  by  means  of  a  feebly  alkaline 
silver  solution,  in  thin  microtome  sections. 


16.  The  Reactions  of  the  Various  Cell-constituents. 

422.  In  many  investigations  it  is  of  interest  to  determine 
what  reaction  the  various  constituents  of  the  cell,  especially 
the  cytoplasm  and  the  cell-sap,  have  in  the  living  cell.  The 
reactions  of  the  sap  pressed  from  large  pieces  of  tissue 
give  very  uncertain  results  in  this  respect,  in  view  of  the 
extensive  division  of  labor  within  the  cell-organism  ;  and  I 
therefore  refrain  from  discussing  further  observations  made 
in  this  way. 

423.  So  far  as  the  cell-sap  is  concerned,  its  reaction  may 
be  directly  determined  in  those  cases  in  which  it  contains 
a  coloring  matter  in  solution  which  changes  its  color  with 
the  reaction.  Such  a  coloring  matter  is  the  so-called  antho- 
cyanin  (§  184),  which  appears  red  when  the  reaction  is  acid, 
blue  when  feebly  alkaline,  and  green  to  yellow  when  strongly 
alkaline.  The  cell-sap  is  generally  alkaline  or  neutral  in 
blue  parts  of  flowers,  but  must  be  acid  in  red  parts.  In  the 
same  cell,  in  the  course  of  its  development,  a  change  in  reac- 
tion may  occur,  as  in  the  flowers  of  Pulmonaria  officinalis, 
which  are  first  red,  and  then  blue.  In  the  last-named  plant, 
as  Pfeffer  has  shown  (V,  140),  a  blue  color  of  the  red  parts 
may  be  produced  at  any  time  by  traces  of  ammonia. 

424.  In  cells  with  colorless  cell-sap,  one  may  reach  conclu- 
sions as  to  its  reaction  by  the  method,  proposed  by  Pfeffer, 
•of  introducing  into  it  an  artificial  coloring  matter  which 
gives  different  colors,  according  to  the  reaction.  According 
to  Pfeffer  (II,  266),  mctJiyl  orange  is  especially  suited  to  this 
purpose,  as  its  orange-yellow  color  is  not  changed  by  dilute 
alkalies.  Pfeffer  used  in  his  experiments  a  .01^  solution. 
Pfeffer  has  (II,  259  and  267)  also  experimented  with  cyanin, 
tropaeolin  and  corallin  ;  but  these  dyes  proved  less  useful. 

425.  It  is,  in  most  cases,  more  difficult  to  determine  the 


SPECIAL    METHODS.  237 

reactions  of  the  protoplasm,  which  never  contains  coloring 
matters  that  show  the  reaction  directly. 

In  the  large  plasmodia  of  ^tJuiliiivi  septicuni  Reinke  (I, 
8)  was  able  to  determine  the  alkaline  reaction  macroscopical- 
ly ;  and  he  deduced  the  presence  of  a  volatile  alkali  from  the 
observation  that  a  bluing  of  litmus-paper  occurred  when  it 
was  not  in  direct  contact  with  the  plasmodium.  But  this 
observation  does  not  in  any  way  exclude  the  presence  of 
other  alkaline  substances  in  the  plasmodium  ;  and  it  has  been 
shown  to  be  probable,  by  Schwarz  (I,  33),  that  the  alkaline 
reaction  of  the  protoplasm  in  the  higher  plants  probably 
does  not  depend  on  the  presence  of  ammonia  or  ammonium 
compounds. 

426.  In  these  plants  Schwarz  (I,  20)  attempted  to  deter- 
mine the  reaction  of  the  protoplasm  by  killing  cells  that 
naturally  have  a  colored  cell-sap  by  alcohol,  heat,  crushing, 
or  electricity,  and  then  noting  the  color  assumed  by  the  dead 
protoplasm.  Again,  he  treated  colorless  cells  in  the  same 
way  in  a  feebly  acidified  extract  of  borecole  leaves  which  is 
yellow-red,  purple-red,  or  red-violet  in  an  acid  solution, 
violet  when  neutral,  and  blue,  blue-green,  grass-green,  yellow, 
or  yellowish  orange  when  feebly  alkaline.  Schwarz  found 
that,  after  killing  by  electricity,  the  protoplasm  of  a  few 
cells  was  blue-green  ;  but  in  most,  it  was  blue-violet,  or  red- 
violet,  and  he  therefore  concluded  that  the  reaction  of  the 
protoplasm  is  alkaline. 

On  the  other  hand,  Arthur  Meyer  (III)  observed  that  the 
coloring  matter  of  kale  is  not  violet,  but  blue,  when  the  re- 
action is  neutral,  and  that,  when  tin-foil  electrodes  are  used 
in  conducting  the  current,  a  violet  tin  compound  of  the  col- 
oring matter  is  formed  and  taken  up  by  the  dead  protoplasm, 
and  that  various  colorations  of  the  extract  may  be  caused 
near  the  electrodes  by  the  decomposition  of  the  salts  con- 
tained in  it.  There  can  therefore  be  no  doubt  that  Schwarz's 
method  cannot  give  trustworthy  results. 

427.  The  most  certain  conclusions  may  be  reached  by 
Pfeffer's  methods  of  introducing  artificially  certain  colored 


238  BOTANICAL   MICROTECHNIQUE.  ^^^^ 

indicators  into  the  living  cell.  In  fact,  Pfeffer  has  (II,  259 
and  266)  already  shown  the  alkaline  reaction  of  the  cytoplasm 
in  various  cells  by  the  aid  of  cyanin  and  methyl  orange.  j 

17.  Plasmolysis  (Plasma-membranes).  ^H 

428.  If  living  plant-cells  are  placed  in  a  solution  of  salt  or, 
the  like,  the  protoplasm  withdraws  from  the  wall  in  the  form 
of  a  continuous  sac,  if  the  solution  be  above  a  certain 
degree  of  concentration,  in  consequence  of  its  power  of 
taking  up  water.  This  proceeding,  now  generally  known  as 
plasmolysis,  offers  the  best  means  of  showing  the  continuity 
of  the  protoplasmic  body  in  cells  poor  in  protoplasm,  and  it 
also  plays  an  important  part  in  various  morphological  and 
physiological  researches. 

429.  Concerning  the  media  used  in  plasmolysis,  it  is 
especially  important  that  they  shall  exert  no  unfavorable 
influence  on  the  cells  in  the  degree  of  concentration  in  which 
they  are  employed.  They  should  also  have  neither  a 
markedly  acid  nor  an  alkaline  reaction.  It  is  best,  then,  to 
use  neutral  salts  like  saltpeter  (KNO3)  or  salt  (NaCl),  or 
organic  compounds  like  cane-sugar  or  glycerine.  The  latter 
has  the  advantage,  in  some  cases,  of  exerting  a  clearing 
effect  in  consequence  of  its  higher  refractive  index.  No 
general  directions  can  be  given  as  to  the  concentration  of 
the  solutions  to  be  used,  and  in  the  choice  of  the  proper 
concentration  the  isotonic  coefficient  "^  of  the  compound  used 
should  be  especially  regarded.  But,  in  general,  clearly  visi- 
ble plasmolysis  may  be  obtained  by  using  a  4^  solution  of 
saltpeter  or  a  15^   solution  of  cane-sugar. 

430.  If  the  objects  to  be  plasmolyzed  are  not  prescribed 
by  the  nature  of  the  investigation,  it  is  best  to  choose  such 
objects  as  are  most  adapted  to  the  observation  of  plasmoly- 
sis, and  as  have  the  greatest  power  of  resisting  the  injurious 
influences  connected  with  their  preparation.  Thus  the  cells 
of  Spirogyra  furnish  very  suitable  material  for  the  demon- 
stration of  plasmolysis.     Such  cells  as  naturally  contain  a 

*  [Cf.  Zimmermann  I,  199-201  :  also  below,  p.  263.] 


SPECIAL   METHODS.  239 

colored  cell-sap  are  also  very  useful,  as,  for  example,  the 
epidermal  cells  of  the  red  lower  surface  of  the  leaf  of  Tra- 
descantia  discolor.  The  earliest  beginnings  of  plasmolysis 
are  easily  observed  in  such  cells. 

431.  In  many  cases  the  plasmolysis  may  be  made  plainer 
by  an  artificial  coloring  of  the  cell-sap.  In  cells  containing 
tannic  acid  this  may  be  accomplished  by  exposing  the  ob- 
jects on  the  slide,  in  a  drop  of  the  plasmolyzing  solution,  to 
the  vapor  of  osinic  acid  for  a  time,  according  to  the  method 
described  in  §  308.  The  protoplasts  are  then  completely 
fixed  in  their  original  position  and  are  much  easier  to  ob- 
serve on  account  of  the  browning  or  blackening  of  the  cell- 
sap.  Plasmolysis  may  also  often  be  made  plainer  by  pre- 
liminary staining  intra  vitam. 

Finally,  by  adding  an  indifferent  pigment,  like  eosin,  to 
the  plasmolyzing  fluid,  a  difference  in  color  between  it  and 
the  cell-sap  may  be  produced. 

To  test  for  the  presence  of  a  living  plasma-body  within 
the  vessels,  Th.  Lange  (I,  404)  injected  large  pieces  of  tissue 
with  a  5^  solution  of  saltpeter  under  the  air-pump,  and 
then  added  a  dilute  solution  of  picric  acid  to  fix  the  tissues. 
After  washing  in  water  and  dehydrating  in  alcohol,  he 
placed  them  in  clove-oil.  Very  good  thin  sections  were 
prepared  from  the  material  so  treated,  and  in  these  the  pro- 
toplasm was  stained  with  borax-carmine  or  eosin. 

Abnormal  Plasmolysis. 

432.  As  was  shown  by  H.  de  Vries  (I),  many  solutions 
cause  the  complete  killing  of  the  protoplasm  and  its  inclu- 
sions, with  the  exception  of  the  inner  plasma-membrane 
bordering  on  the  cell-sap,  which  is  not  changed  in  its 
osmotic  relations,  so  that  it  still  completely  excludes  the 
cell-sap  (cf.  Fig.  60).  If  this  proceeding,  which  de  Vries 
calls  abnormal  plasmolysis,  does  not  justify  such  far-reaching 
conclusions  as  he  has  drawn  from  it  (cf.  on  this  point  Pfeffer 
VIII,  240),  yet  abnormal  plasmolysis  may  do  good  service 
in  many  investigations. 


240 


BO  TA NICA L   MICRO  TECHNIQ  UE. 


of 


433.  To  produce  it,  one  may  use  a  10%  solution  ot  sai 

peter  to  which  a  httle  eosin  h; 
been  added.  This  pigment  ha< 
the  double  advantage  of  at  onc< 
staining  the  killed  part  of  the  pro- 
toplasm and  of  causing  a  sharpei 
definition   of  the  uncolored   cell- 

'"„°o,^arp';l'm.?W»-'r»TSV.oi"sat  sap.     Pretty  large   Spirogyra-ctWi 
S::"h/°mo°?soiateTvi"Soiir'";  furnish  excellent  objects  for  stud 

remains  of  the  dead  chloroplasts.         /    r    p«*        fir>\ 

434.  For  fixing    the  isolated  vacuolar  membranes  Went 
recommends   (I,   314)  ^-i^  chromic  acid,  which  he  allows 
to  act  one  or  two  days.     He  then  washes  the  objects  in 
running  water  six   hours   and  transfers  them,  in    the  usual 
way,  to  parafifine,  for  the  preparation  of  microtome  sections 


\ 


18.  Methods  of  determining  whether  certain  Bodies  lie  In  the  Cyt 
plasm  or  in  the  Cell-sap. 

435.  This  question  often  cannot  be  answered  by  direct 
microscopical  examination,  and  already  various  methods 
have  been  devised  which  make  a  certain  decision  possible 
even  in  diflficult  cases.  Wakker  (I)  observed,  with  micro- 
scope tipped  down,  what  motions  the  bodies  in  question  un- 
derwent with  the  slide  in  a  vertical  position.  If  they  simply 
fall  downward  in  the  cells  in  a  vertical  line  in  consequence 
of  their  greater  specific  gravity,  it  is  very  probable  that  the 
bodies  lie  in  the  cell-sap.  But,  since  the  starch-bearing  chro- 
matophores  of  the  starch-sheath,  which  undoubtedly  lie  in 
the  cytoplasm,  sink  down  rather  rapidly  in  the  cells,  as  was 
recognized  by  Dehnecke  (I,  9)  and  Heine  (I,  190),  it  has 
seemed  useful  to  me  to  modify  this  method  by  reducing  the 
ready  displaceableness  of  the  protoplasm  by  killing  the 
cells,  which  may  be  easily  done  with  an  iodine  and  potas- 
sium-iodide solution.  The  movement  of  the  chromato- 
phores  in  the  starch-sheath  was  at  once  stopped  by  iodine 
solution,  while  the  protein  crystalloids  in  the  epidermis  of  the 
leaf  of  Polypodium  irreoides,  which  undoubtedly  lie  in  the 


SPECIAL   METHODS.  24! 

cell-sap,  continued  the  motions  due  to  their  weight  after  the 
same  treatment  (cf.  Zimmermann  II,  68). 

436.  Wakker  (I)  has  also  used  abnormal  plasmolysis  with 
a  loio  solution  of  saltpeter,  containing  eosin  (cf.  §§  432  and 
433),  for  the  same  purpose.  It  is  often  to  be  determined 
with  certainty  by  direct  observation  whether  the  bodies  in 
question  lie  in  the  vacuoles  isolated  by  this  method.  Be- 
sides, a  further  confirmation  of  the  conclusions  reached  by 
direct  observation  may  be  obtained  from  the  movements 
taking  place  in  such  preparations  when  the  slide  is  placed 
vertically. 


19.  Aggregation. 

437.  Complicated  changes  of  arrangement  take  place 
within  the  cells  of  the  glandular  hairs  of  Drosera  rotundi- 
folia  in  consequence  of  chemical  stimulation,  which  consist 
essentially  in  the  origin  of  rapid  circulation-currents  in  the 
protoplasm  and  the  breaking  up  of  the  large  central  vacuole 
into  a  large  number  of  small  vacuoles  which  gradually  con- 
tract more  and  more.  This  process,  discovered  by  Darwin 
and  studied  in  detail  especially  by  H.  de  Vries  (II),  will  be 
termed  exclusively,  in  the  following  account,  aggregation, 
in  agreement  with  de  Vries,  though  Darwin  and  various 
other  authors  use  the  same  term  also  for  the  artificial  pre- 
cipitation which  accompanies  the  process,  but  also  occurs  in 
very  many  plants. 

438.  The  tentacles  of  Drosera  are  especially  adapted  to 
the  observation  of  aggregation,  since  their  vacuoles  stand 
out  very  sharply  on  account  of  their  red  cell-sap.  Aggre- 
gation may  be  produced  in  them  by  bringing  a  leaf  of  the 
plant  in  contact  with  an  insect,  a  bit  of  cooked  albumen,  or 
the  like.  It  may  also  be  observed  in  isolated  tentacles 
which  have  been  placed  in  a  i^  solution  of  ammonium  car- 
bonate. Aggregation  then  begins  at  the  bases  and  at  the 
tips  of  the  tentacles,  and  may  be  especially  well  followed  in 
its  separate  stages  at  their  middles  (cf.  Fig.  61). 

439.  According  to  recent  investigations  of  Bokorny  (IV), 


■42 


B O  TA  XICA  L   MICR O  TE  CHNIQ  UE. 


lo  observed  aggregation  in  other  plants  also,  it  is  produced 
I  J  J  J  jj         by  the  most  various  <^^j/r'substances ; 

but  if  they  have  highly  poisonous 
properties,  as  caustic  potash  or  am- 
monia, they  must  be  used  in  very 
dilute  form.  This  author  observed 
a  very  far-reaching  aggregation  on 
leaving  superficial  sections  from  peri- 
anth-leaves of  Ttilipa  suaveolens  for 
tv^o  or  three  days  in  a  .i^  coffein  so- 
lution. 
440.  A  somewhat  different  appear- 

FiG.  61.— Cell  from  a  marginal  ten-  ,   .    ,     .  .     ,  ,1 

lacie  of  the  leaf  of  Dresera  ro-  ance,  whicli  IS  Certainly  Very  closely 

tundifolin     in     three     different  ,        -^  ^ 

stages  of  aggregation,  caused  related  to  acfgrecration,  was  observed 

by  a  .ijf  solution  of  ammonium  '^^      ° 

carbonate.      The     interval     be-    by  Bokomy  (I  V,  45  I  )  On  placing^  SCC- 

tween  I  and  II  is  six  minutes,       ''  j    \        ^  -tj    j  r  & 

and  that  between  II  and  III,  tions  of  the  maig^in  of  the  stigma  of 

two     minutes.      After     H.     de  o  t> 

vries.  Ctocus  vcrmis  in  a  .i^   solution   of 

coffein.  The  vacuolar  wall  did  not  draw  together  as  in 
normal  aggregation,  but  the  whole  protoplasmic  mass  con- 
tracted, so  that  there  arose  between  it  and  the  cell-mem- 
brane a  space  filled  with  water. 

20.  Artificial  Precipitates. 

441.  Artificial  precipitates  may  be  produced  in  the  cyto- 
plasm and  in  the  cell-sap  without  injury  to  the  vitality  of 
the  cell,  by  the  most  various  reagents.  Such  excretions  may 
often  arise  in  the  cell-sap  during  plasmolysis.  The  chemical 
composition  of  these  bodies  has  as  yet  been  determined  in 
but  few  cases ;  but  it  may  be  regarded  as  very  probable  that 
protein-\\}^^  compounds,  especially,  are  widely  distributed  in 
these  precipitates  with  tannins  and  related  substances. 

442.  Precipitates  which  consist  of  relatively  pure  tannic 
acid  are  produced  in  a  great  variety  of  cells  by  alkaline  car- 
bonates (cf.  §  206).  That  these  usually  globular  bodies  con- 
tain no  important  amount  of  proteid  substances  was  recog- 
nized by  Klercker  (I). 

Similar  precipitates  were  produced  by  Bokorny  (V  and 
IV)  in  various  plant-cells  by  the  most  various  basic  com- 


SPECIAL   METHODS. 


243 


pounds,  as,  for  example,  by  a  .1^  solution  of  coffein.  These 
may  lie  partly  in  the  cytoplasm  and  partly  in  the  cell-sap, 
and,  since  they  also  occur  in  cells  free  from  tannin,  cannot 
consist  of  tannin  in  all  cases,  at  least.  But  to  what  extent 
proteid  substances  or  "■  active  albumen  "  (of.  §  445),  which 
forms  their  chief  constituent,  according  to  Bokorny,  occur 
in  them  cannot  be  certainly  determined  from  our  present 
Icnowledge.  It  is  not  impossible  that  we  have  here  to  do 
-with  very  various  bodies. 

443.  Precipitates  occurring  in  plasmolysis  were  first 
observed  by  PfefTer  (II,  245)  in  the  root-hairs  of  Azolla,  and 
they  occurred  in  the  same  manner  whether  the  plasmolysis 
was  accomplished  by  means  of  sugar,  saltpeter,  or  calcium 
<:hloride.  These  precipitates  agree  essentially  with  those 
produced  by  ammonium  carbonate,  and  consist  chiefly 
of  tannin,  in  most  cases.  But,  as  af  Klercker  (I,  29)  has 
shown,  other  substances  must  be  present  in  the  cell-sap 
"which  remain  dissolved  in  the  vacuolar  fluid  during  the 
■excretion,  and  prevent  the  partial  re-solution  of  the.  tannin. 
It  is  also  noteworthy  that  Klercker  (I,  43)  has  observed  similar 
precipitates  in  artificial  cells  of  tannate  of  glue,  on  plasmo- 
lyzing  them  with  a  solution  of  saltpeter,  while  plasmolysis 
with  sugar  contracted  the  whole  cell  into  a  glassy  mass. 

444.  Plasmolytic  precipitates  which  are  not  due  to  the 
presence  of  tannins  may  be  very  easily  ob- 
served in  the  epidermal  cells  of  the  red  under 
surface  of  the  leaves  of  Tradesca?itia  discolor. 
If  tangential  sections  of  these  cells  be  placed  in 
a.  small  dish  with  a  10^  solution  of  saltpeter,  it 
may  be  seen  in  ten  minutes  that  strong  plasmo- 
lysis has  taken  place  in  all  the  cells,  and  pretty 
strongly  refractive,  deeply  colored  spheres  (cf.  p,G.  62.-Epider- 
Fig.  62)  occur  in  most  cells,  while  the  cell-sap  Sl^unTJr^ide"" 
has  in  some  cases  become  markedly  clearer.  ^^scantiadiscoiTr 
If  water  is  afterwards  added,  these  precipitates     J  ^^^  ^s^aitpeter 

,.         ,         ,         «T  •     •.     ,  1  1        solution,      show- 

are  redissolved.     No  precipitates  are  produced      ing   piasmoiytic 

in  these  cells  by  ammonium  carbonate,  and  no      p^'^'^'P''-^  ^^• 
blackening  occurs  with  osmic  acid. 


244  BOTANIC  A. 

21.  The  Loew-Bokorny  Reagent  for  "Active  Albumen." 

445.  Loew  and  Bokorny  (cf.  I  and  II)  have,  in  various 
papers,  maintained  the  view  that  living  and  dead  proto- 
plasm  differ  from  each  other  in  that  only  the  former  h:ij 
the  power  to  precipitate  silver  from  an  alkaline  silver- 
solution.  These  authors  conclude  from  this  that  livinj 
albumen  contains  aldehyde  groups  which  at  once  underg( 
a  breaking  up,  at  its  death.  [They  have  observed  a  pre- 
cipitation, in  living  cells  treated  with  a  .1^  solution  oi 
coffein,  of  abundant  small  granules  which  they  believe  to^| 
consist  of '*  active  albumen,"  and  term  '*proteosomes."j 

446.  It  has,  however,  been  shown  by  various  authors  (cf.j 
especially  Pfeffer  VII)  that  the  observations  of  Loew  and 
Bokorny  are  partly  incorrect  and  that  the  bodies  called  by 
them  "  active  albumen  "  certainly  consist  in  part  of  tannin 
and  similar  substances.  But,  since  the  Loew-Bokorny 
"  Life-reagent  "  has  already  been  used  by  other  investigators 
for  the  recognition  of  living  albumen,  and  may  perhaps  be 
capable  of  furnishing  a  basis  for  some  conclusions  concern- 
ing the  contents  of  the  cell,  when  more  critically  employed^ 
the  methods  used  by  these  investigators  may  be  briefly  out- 
lined here. 

447.  The  silver  reagent  called  by  Loew  and  Bokorny 
"  Solution  A  "  is  prepared  by  mixing  13  ccm.  of  caustic 
potash  solution  of  specific  gravity  1.33  (containing  33 J^  of 
KOH)  and  10  ccm.  of  aqua  ammonia  of  specific  gravity  .96 
(containing  9^  of  NH3)  and  diluting  the  mixture  to  icx> 
ccm.  For  use,  i  ccm.  of  this  solution  is  mixed  with  i  ccm. 
of  a  i^  silver  nitrate  solution,  and  the  mixture  is  diluted 
to  one  liter  (1000  ccm.). 

448.  The  "  Solution  B  "  is  prepared  by  adding  5  to  10  ccm, 
of  a  saturated  solution  of  lime  to  a  liter  of.  a  j^Vo^  solution 
of  silver  nitrate. 

Both  solutions  must  be  used  in  large  quantities  on  account 
of  their  extreme  dilution,  and  but  a  small  number  of  the 
objects  used  should  be  placed  in  them.  The  deposition  of 
silver  usually  begins  only  after  some  hours,  and  it  is  gen- 


SPECIAL    METHODS.  245; 

erally  necessary  to  leave   the   objects   from   5  to  24  hours- 
in  the  solutions. 

A  more  rapid  reaction  is  obtained  with  a  more  concen-^ 
trated  solution  containing  i  gram  AgNO,  and  .3  gram  NH, 
to  a  liter  of  water.  This  can  be  used,  however,  only  with 
resistant  objects  and  with  such  as  contain  neither  sugar  nor 
tannin  (cf.  Bokorny  VI,  195). 

22.   Protoplasmic  Connections. 

449.  Proof  has  been  furnished  by  the  investigations  of 
Tangl,  Gardiner,  Russow,  and  others  that,  besides  the  ele- 
ments of  the  sieve-tubes,  the  protoplasts  of  the  separate: 
cells  of  various  other  tissue-systems  are  in  direct  connection, 
with  each  other  through  perforations  of  their  cell-walls.. 
Kienitz-Gerloff  (I,  22)  has  recently  concluded  from  his  re- 
searches that  all  the  living  elements  of  the  entire  body  of 
the  higher  plants  are  united  by  protoplasmic  threads.  The- 
threads  which  accomplish  this  union,  the  so-called  proto- 
plasmic connections,  are,  however,  in  most  cases  so  fine  that 
nothing  can  be  seen  of  them  within  the  living  cell ;  and 
rather  complicated  preparation-methods  are  necessary  to- 
their  recognition. 

450.  If  one  has  to  do  with  relatively  tJiick  protoplasmic 
connections,  like  those  of  many  sieve-tubes,  they  may  be 
made  visible,  in  many  cases,  simply  by  treatment  with 
chloroiodide  of  zinc  or  with  iodine  and  sulphuric  acid- 
Gardiner  (II,  55,  note  4)  recommended  for  this  purpose  a_ 
solution  of  Hofmann's  violet  in  concentrated  sulphuric  acid. 
This  solution  has  a  brownish  color  and  does  not  stain  strongly 
sections  placed  in  it.  But  if  the  acid  is  washed  out  with  a 
large  quantity  of  water,  after  acting  for  about  half  a  minute,, 
the  sections  take  at  first  a  green,  then  a  bluish,  and  finally  a 
violet  color,  and  the  protoplasts  are  almost  exclusively 
colored.  I  have  obtained  in  this  way  very  instructive 
preparations  of  alcoholic  material,  adding  to  pretty  thin 
sections  on  the  slide,  after  drying  them  externally  with  filter- 
paper,  a  drop  of  the  sulphuric  acid  containing  the   staining- 


246  BOTANICAL   MICROTECHNIQUE. 

material,  and  at  once  dropping  on  a  cover-glass  to  prevent 
too  great  warping  of  the  sections.  After  a  short  time  the 
whole  slide  is  plunged  into  a  large  vessel  of  v^^ater  in  which 
it  remains  until  the  sections  have  beconne  clear  violet.  Al- 
though the  cover-glass  usually  separates  from  the  slide,  some 
sections  generally  remain  attached  to  it,  and  these  can, 
naturally,  best  be  used  for  study. 

451.  A  very  deep  staining  of  the  protoplasmic  connections 
contained  in  the  sieve-pores  may  be  obtained  in  microtome 
sections  of  stems  of  Cuairbitacece  by  the  use  of  Altmann's 
acid  f  uchsin  method  (cf.  §  345).  This  brings  out  the  proto- 
plasmic threads  without  the  previous  use  of  any  swelling 
media. 

In  many  cases  the  double  staining,  described  in  §  290, 
Avith  aniline  blue  and  eosin  gives  very  fine  preparations  of 
the  sieve-plates  with  the  protoplasmic  connections  passing 
through  them. 

452.  Delicate  protoplasmic  connections,  on  the  other  hand, 
have  as  yet  been  made  visible  only  by  the  successive  action 
of  swelling  and  staining  media. 

For  swelHng,  chloroTodide  of  zinc  and  sulphuric  acid  have 
been  chiefly  used. 

ChloroTodide  of  zinc  was  recommended  especially  by 
Oardiner  (II).  He  treated  sections  of  fresh  material  first 
with  a  solution  of  iodine  and  potassium  iodide,  then  added 
chloroTodide  of  zinc  and  let  it  act  a  longer  or  shorter  time, 
according  to  the  capacity  of  the  membranes  for  swelling, 
commonly  about  12  hours.  Before  staining,  the  chloroTodide 
was  washed  out  with  water  or,  where  the  membranes  were 
much  swollen,  with  alcohol. 

453.  When  sulphuric  acid  is  used,  the  sections  are  also 
usually  first  fixed  with  iodine  and  potassium  iodide  solution. 
Kienitz-Gerloff  (I,  8)  used  for  this  purpose  a  solution  con- 
taining .05  gram  of  iodine  and  .20  gram  of  potassium  iodide 
in  15  grams  of  water.  He  recommends,  especially  for  juicy 
tissues,  the  method  used  by  A.  Fischer  (III)  with  the  best 
results  in  the  study  of  the  contents  of  the  sieve-tubes  (cf.  § 
455).     This   author   scalded    large  plants   or  large   parts  of 


SPECIAL    METHODS.  247 

plants  in  boiling  water  as  quickly  as  possible  and  hardened 
them,  before  cutting,  in  absolute  alcohol. 

Concerning  the  strength  of  the  sulphuric  acid  used  for 
swelling  it  may  be  remarked  that  it  has  been  used  partly  in 
concentrated  form,  partly  after  dilution  with  one  fourth  its 
volume  of  water.  With  membranes  which  swell  strongly,, 
the  dilute  acid  is,  of  course,  to  be  preferred.  The  time  for 
it  to  act  depends  chiefly  on  the  character  of  the  membranes. 
But  usually  a  few  seconds  is  sufficient  for  concentrated  acid. 
The  acid  must,  of  course,  be  removed  by  careful  washing  in 
water,  before  staining. 

453.  The  following  dyes  have  been  used  for  staining  sec- 
tions  treated  in  this  way. 

1.  Hofmanns  blue  (same  as  aniline  blue).  Gardiner  (II) 
recommends  a  solution  of  this  dye  in  50^  alcohol  saturated 
with  picric  acid.  The  solution  is  washed  out  with  water^ 
and  the  preparation  is  then  either  mounted  in  glycerine,  or 
is  gradually  transferred  through  dilute  to  strong  alcohol, 
cleared  with  clove-oil,  and  mounted  in  Canada  balsam.  In 
this  way  very  permanent  preparations  of  the  protoplasmic 
connections  may  be  obtained. 

Gardiner  also  used  a  solution  of  Hofmann's  blue  in  50^ 
alcohol  acidified  with  a  few  drops  of  acetic  acid.  The  sub- 
sequent treatment  is  the  same  as  in  the  previous  case. 

Terletzki  (I,  455)  stains  simply  with  a  strong  aqueous 
solution  of  aniline  blue  and  examines  the  preparation  in 
water,  after  washing. 

2.  Hofmamis  violet  is  used  by  Gardiner,  simply  in  an 
aqueous  solution.  This  at  first  stains  wall  and  protoplasm 
about  equally ;  but,  after  lying  for  a  long  time  in  glycerine, 
in  many  cases  several  days,  the  color  is  removed  from  the 
walls,  while  the  protoplasts  and  protoplasmic  connections 
remain  strongly  stained. 

3.  Methyl  violet  is  recommended  by  Kienitz-Gerloff  (I,  9) 
for  such  cells,  like  those  of  hairs,  etc.,  as  do  not  permit  the 
penetration  of  Hofmann's  blue,  on  account  of  cuticulariza- 
tion  of  the  walls.  He  uses  it  in  a  concentrated  aqueous 
solution. 


248 


BO  TA  NIC  A  L   MICRO  TECHNIQ  UE. 


It  may  be  observed  that  Gardiner  finds  it  useful  to  brush 
over  the  sections  with  a  camel's-hair  brush  after  swelling 
^nd  staining. 

454.  A  method  differing  essentially  from  previous  ones  has 
lately  been  used  by  Kohl  (I,  12)  for  demonstrating  the  proto- 
plasmic connections.  It  agrees  in  essence  with  Loefifler's 
method  for  staining  cilia  (§  476).  After  preliminary  mor- 
•danting  with  tannin,  Kohl  stained  with  nietJiyle7ie  blue  or 
Bismarck  brown  ;  and  if  the  staining  of  cell-walls  or  gelatin- 
ous sheaths,  due  to  the  presence  of  pectic  substances,  inter- 
fered with  observation,  these  substances  were  removed  by 
an  acid.  This  author  has  not  given  more  exact  details  of 
iiis  method. 

23.  Contents  of  Sieve-tubes. 

455.  Since  the  contents  of  the  sieve-tubes,  which  com- 
municate by  relatively  large  openings,  are  partly  pressed 
out  on  cutting  the  tissues,  and  undergo  the  most  various 
changes,  A.  Fischer  (III)  has  devised  a  method  of  fixing  the 


r<~i^  J  yy 


Fig.  63.--Parts  of  sieve-tubes  of  Cucurbita  Pefio.    a,  from  a  plant  cut  and  then  scalded: 
X,  '  Schlauchkopf  .  b,  scalded  in  an  uninjured  condition  (X  675).     After  Fischer. 

-contents  of  the  sieve-tubes  in  the  uninjured  tissues.  For 
this  purpose  he  plunges  carefully  unpotted  plants  or  pieces 
of  an  uninjured  plant,  such  as  branch-tips,  into  boiling  water 
and  leaves  them  in  it  until  the  contents  of  the  sieve-tubes 
are  coagulated,  for  which  two  to  five  minutes'  exposure  is 
usually  quite  sufificient.  He  was  able  to  show  that  the 
accumulations  of  strongly  refractive  and  readily  staining 
substance  observed  at  one  side  of  the  sieve-plate  in  Cucur- 


SPECIAL   METHODS.  249 

bita  and  many  other  plants  when  the  ordinary  methods  are 
employed,  the  so-called  "  Schlauchkopfe "  (Fig.  63,  a^  j), 
represent  artificial  products  and  originate  only  when  the 
sieve-tube  system  is  cut.  They  were  entirely  wanting  in 
material  scalded  as  above  (Fig.  63,  U)y  but  were  not  changed 
by  boiling  water  in  tissues  where  they  had  arisen  from  cut- 
ting before  the  scalding. 

456.  According  to  investigations  of  this  scalded  material 
by  A.  Fischer  (VI),  we  may  distinguish  three  sorts  of  sieve- 
tubes,  according  to  the  organization  of  their  contents,  as 
follows : 

1.  Sieve-tubes  with  coagulable  contents  (in  the  Cucurbi- 
taced)  have  a  thin  protoplasmic  wall-layer  and  a  clear  sap 
coagulable  by  heat. 

2.  Sieve-tubes  with  mucus  (as  in  Humulus)  contain  a 
delicate  wall-layer  filled  with  large  and  small  slime-masses 
and  a  clear,  non-coagulable,  watery  fluid. 

3.  Sieve-tubes  with  starch-grains  (as  in  Coleus)  contain  a 
delicate  wall-layer  carrying  small  masses  of  mucus  and 
a  clear,  non-coagulable  fluid  with  small  starch-grains. 


METHODS  OF  INVESTIGATION  FOR  BACTERIA. 

457.  Since  the  methods  employed  in  Bacteriology  differ 
in  many  respects  from  those  used  in  the  study  of  other 
plants,  I  have  preferred  to  collect  the  former  into  a  special 
chapter.  In  the  following  pages  I  have  not  attempted  to 
give  an  even  approximately  exhaustive  compilation  of  bac- 
teriological methods.  I  have  rather  chosen  to  bring  together 
a  number  of  trustworthy  methods  of  preparation  which 
should  be  quite  sufficient  for  most  cases  in  the  study  of 
Bacteria.  I  must  refer  persons  who  wish  to  devote  them- 
selves especially  to  Bacteria  to  the  special  works  on  the 
subject,  particularly  those  of  Giinther  (I)  and  Hueppe  (I). 

I.  The  Observation  of  Living  Bacteria. 

458.  The  observation  of  living  Bacteria  may  be  generally 
conducted  like  that  of  other  lower  organisms.  If  it  is  to  be 
continued  for  a  long  time,  the  Bacteria  may  best  be  placed 
in  a  hanging  drop  (cf.  §  2).  Under  some  circumstances,  a 
frequent  renewal  of  the  culture-fluid  is  necessary. 

In  case  of  rapidly  motile  Bacteria,  they  may  be  brought 
to  rest  by  proper  fixing  media,  like  the  fumes  of  iodine  or 
of  osmic  acid. 

Finally,  I  may  remark  that,  in  some  cases,  the  dark-field 
illumination  of  the  Abb^  condenser  may  be  used  with  suc- 
cess in  the  examination  of  Bacteria. 

250 


BACTERIA.  2c;i 


II.  Fixing  Methods. 
1.  Cover-glass  Preparations. 

a.    Fixing  by  Dry  Heat. 

459.  For  fixing  Bacteria  from  fluids  containing  them, 
from  gelatine  cultures,  or  the  like,  dry  heat  is  almost 
exclusively  used  at  present,  and  commonly,  according  to 
R.  Koch's  method,  as  follows  : 

A  small  drop  of  fluid  containing  Bacteria  is  transferred, 
by  means  of  a  platinum  wire  sterilized  by  heat,  to  a  carefully 
cleaned  cover-glass,*  and  spread  out  over  it  as  evenly  as 
possible.  If  the  Bacteria  are  taken  from  a  solid  substratum, 
a  drop  of  water  is  first  placed  upon  the  cover  and  a  very 
small  particle  of  the  material  is  rubbed  up  in  it  as  completely 
as  possible  with  a  platinum  wire. 

The  fluid  is  now  allowed  to  evaporate  from  the  cover- 
glasses  thus  smeared  with  Bacteria,  at  the  ordinary  tempera- 
ture, until  the  preparation  is  "air-dry." 

The  Bacteria  are  then  fixed  by  passing  the  cover-glasses,, 
with  their  Bacteria-sides  upward,  three  times  through  the 
non-luminous  flame  of  a  Bunsen  burner.f  Johne's  rule  may- 
give  an  idea  of  the  rapidity  with  which  this  should  be  done. 
According  to  this,  the  hand  should  describe  an  horizontar 
circle  a  foot  in  diameter  in  a  second,  moving  at  an  equals 
rate  throughout  the  course,  and  passing  through  the  flame: 
at  one  part  of  it. 

460.  In  this  way  the  Bacteria  are  so  fastened  to  the  cover- 
glass  that  they  may  be  treated  with  staining-fluids  and  other 
media  without  fear  of  separation.  It  is  thus  possible  to 
stain  or  restain,  at  any  time,  preparations  which  have  been 
mounted  for  a  long  time  in  Canada  balsam.     For  this  pur- 

*The  cleaning  of  cover-glasses  may  be  accomplished  by  the  method  pro- 
posed by  Giinther  (I,  40,  note),  which  consists  in  heating  the  glasses,  after 
cleaning  with  alcohol,  in  the  non-luminous  flame  of  a  Bunsen  burner. 

f  In  these  and  the  following  manipulations  the  so-called  Kuehne  forceps 
are  very  convenient.  Their  arms  are  bent  at  about  i^  cm.  from  their  tips^ 
and  end  in  broad  surfaces.  . 


252  BOTANICAL   MICROTECHNIQUE. 

pose  one  need  only  gently  warm  the  preparation  until  the 
balsam  becomes  fluid,  remove  the  balsam  from  the  raised 
cover-glass  with  xylol,  and  finally  wash  off  the  xylol  with 
alcohol. 

It  may  be  remarked,  finally,  that  Bacteria  fixed  in  the 
above  manner  may  be  preserved  in  this  condition  for  an 
indefinite  time  without  harm,  if  they  are  protected  from 
dust  and  moisture  by  being  wrapped,  for  example,  in  filter- 
paper. 

461.  To  remove  the  strongly  staining  substance  from  the 
red  blood-corpuscles,  in  preparations  from  the  blood,  Gun- 
ther  (I,  63)  recommends  rinsing  the  objects,  fixed  in  the 
usual  way,  in  1-5^  acetic  acid.  The  stainable  haemoglo- 
bin is  thus  extracted  from  the  red  corpuscles,  and  a  large 
part  of  the  blood-plasma  is  washed  out  of  the  preparations, 
leaving  the  Bacteria  unchanged.  Preparations  which  give 
no  satisfactory  results  with  this  method,  on  account  of  hav- 
ing been  kept  dry  for  a  long  time,  have  been  treated  by 
Gunther  with  a  2-yf>  solution  of  pepsin,  with  the  best  re- 
sults. 

b.  Other  Fixing  Methods. 

462.  Since  certain  inequalities  can  hardly  be  avoided  with 
the  fixing  methods  described  above,  H.  Moeller  (II,  274) 
has  proposed  fixing  the  air-dry  preparations  with  absolute 
alcohol,  instead  of  heating  them  ;  he  leaves  them  in  this  fluid 
two  minutes. 

463.  A.  Fischer  (II)  demonstrated  the  noteworthy  fact 
that  artificial  appearances  often  arise,  especially  when  prepa- 
rations are  allowed  to  dry,  which  are  chiefly  the  result  of 
plasmolysis  of  the  bacterial  cells  (cf.  §  428).  As  Fischer  has 
shown,  the  Bacteria  are  plasmolyzed  by  solutions  of  pretty 
slight  concentration.  In  general  a  i^  salt  solution  is  suffi- 
cient to  produce  plasmolysis  in  most  Bacteria.  Fischer  (II, 
73)  recommends  the  use  of  a  loj^  solution  of  lactic  acid  for 
fixing  Bacteria,  which  does  not  prevent  subsequent  staining 
with  alcoholic  solutions  of  aniline  dyes. 

464.  Besides,  the  fixing  methods  used  for  higher  plants 


BACTERIA.  253 

may  certainly  be  used  with  some  success  in  the  investiga- 
tion of  the  minute  structure  of  the  bacterial  cell.  Their 
use  may  generally  be  successfully  carried  out  by  Overton's 
method.  But  it  should  be  noted  that  the  membranes  of  the 
Bacteria  are  characterized  by  relatively  great  impermeabil- 
ity. It  has  been  shown  by  A.  Fischer  (II,  72)  that  i^  osmic 
acid  and  a  i^  solution  of  corrosive  sublimate,  in  particular, 
cause  only  an  incomplete  fixation  of  bacterial  cells. 

2.  Sections. 

465.  Absolute  alcohol  is  commonly  used  for  fixing  Bac- 
teria within  infected  organisms,  pieces  of  the  tissue  being 
placed  directly  in  it.  The  microtome  should  be  used  for 
cutting  sections  from  these,  after  imbedding  in  paraffine 
(§  43)  or  celloidin  (§  49a). 


III.  Staining  Methods. 

466.  Under  this  head  a  number  of  methods  will  be  de- 
scribed which  can  be  successfully  used,  in  most  cases,  for 
recognizing  the  presence  of  Bacteria  in  a  fluid  or  in  a  dis- 
eased organism.  Then  follows  a  special  description  of  stain- 
ing methods  for  tubercle  Bacilli,  the  spores,  and  the  cilia  of 
Bacteria. 

I.  Staining  with  Loeffler's  Methylene  Blue. 

467.  Loefifler's  methylene  blue  consists  of  30  ccm.  of  a 
concentrated  alcoholic  solution  of  methylene  blue  and  100 
ccm.  of  an  aqueous  .01^  solution  of  caustic  potash.  It  keeps 
indefinitely. 

With  cover-glass  preparations,  it  may  be  used  by  care- 
fully warming  the  preparation  with  a  few  drops  of  the  stain 
until  steam  is  seen  to  rise,  then  washing  off  the  stain  with 
water  and  drying  in  the  air,  without  heat.  A  drop  of  Cana- 
da balsam  is  then  placed  on  a  slide,  and  the  cover-glass  is 
placed  on  it.  This  method  does  not  give  a  very  deep  stain, 
but  often  brings  out  delicate  differentiations  sharply. 


254  BOTANICAL   MICROTECHNIQUE. 

For  staining  sections  which  would  be  injured  by  warnning^ 
the  methylene  blue  may  be  allowed  to  act  for  a  longer 
time.  The  staining  fluid  is  then  washed  off  with  water,  and 
the  preparation  is  transferred  to  balsam,  either  by  being  first 
allowed  to  dry  (§  23),  or  by  the  use  of  aniline  between  water 
and  xylol  (§  24).  Many  Bacteria,  such  as  the  anthrax  Ba- 
cillus, endure  treatment  with  alcohol  very  well,  and  may 
therefore  be  transferred  to  balsam  in  the  ordinary  way. 


2.  Ziel's  Carbol-fuchsin. 

468.  Ziel's  solution  of  carbol-fuchsin  is  prepared  by  rub- 
bing up  one  gram  of  fuchsin  with  100  ccm.  of  a  5^  aqueous 
solution  of  carbolic  acid,  with  the  gradual  addition  of  10 
ccm.  of  alcohol.     It  is  very  stable. 

With  cover-glass  preparations,  it  is  allowed  to  act  only 
about  a  minute.  Under  some  circumstances  its  action  may 
be  hastened  by  warming.  The  preparations  may  be  washed 
in  water  and  mounted,  after  drying,  in  Canada  balsam.  But 
strongly  stained  objects  will  endure  longer  washing  with 
alcohol,  and  may  be  transferred  to  xylol,  and  then  to 
Canada  balsam. 

Carbol-fuchsin  seems  less  adapted  to  use  with  sections^ 

3.  Ehrlich's  Aniline-water  Solutions. 

469.  These  solutions  are  prepared  by  adding  1 1  ccm.  of  a 
concentrated  alcoholic  solution  of  fuchsin,  gentian  violet,  or 
methyl  violet  to  100  ccm.  of  aniline-water.  Turbidity  arises 
at  first  in  this  mixture,  which  prevents  its  immediate  use. 
But  it  may  be  used  for  staining  in  24  hours,  after  previous 
filtering.     It  remains  fit  for  use  but  a  few  weeks. 

It  is  sufficient  to  let  these  solutions  act  for  a  minute^ 
while  heated,  on  cover-glass  preparations.  The  dye  is  then 
washed  off  with  water,  and  the  preparation  is  dried  and 
mounted  in  balsam. 

Betteir  staining  of  the  Bacteria  contained  in  sections  is 
obtained  by  Gram's  method,  described  in  the  next  paragraph. 


BACTERIA.  255 


4.  Gram's  Method. 


470.  The  so-called  Gram's  method  is  adapted  especially 
for  sections,  because  it  stains  the  Bacteria  in  them  deeply 
without  staining  the  nuclei  at  the  same  time.  But  it  may 
also  be  used  with  cover-glass  preparations,  especially  if  they 
contain  many  other  stainable  bodies  besides  Bacteria. 

According  to  Gram's  original  account,  this  method  con- 
sisted in  placing  the  sections  first  for  several  minutes  in 
Ehrlich's  aniline-water-gentian-violet  solution  (§  469),  and 
then  transferring  them  to  a  solution  containing  one  part  of 
iodine  and  two  parts  of  potassium  iodide  in  300  parts  of 
water.  After  a  few  minutes  in  this,  they  are  washed  with 
alcohol  until  no  more  color  comes  off,  then  transferred  to 
clove-oil,  which  removes  more  of  the  dye,  and  finally  mounted 
in  balsam. 

471.  But,  according  to  Giinther  (I,  89),  it  is  better,  in 
most  cases,  to  treat  the  sections,  after  removal  from  the 
iodine  solution,  for  half  a  minute  with  alcohol,  then/?/5/  ten 
seconds*  with  3^  hydrochloric  acid-alcohol,  and  then  at 
once  with  pure  alcohol  until  they  are  completely  decolor- 
ized. For  transferring  them  from  alcohol  to  balsam,  this 
author  recommends  xylol,  instead  of  clove-oil. 

According  to  the  method  described  by  Weigert,  aniline 
is  gradually  dropped  upon  the  sections,  differentiating  and 
dehydrating  them,  and  they  are  then  passed  through  xylol 
into  balsam. 

472.  A  sharp  double  staining,  by  which  the  nuclei  are 
differently  stained  from  the  Bacteria,  may  be  obtained  by 
preliminary  staining  with  picro-carmine  (§  318).  This  solu- 
tion is  allowed  to  act  one  or  two  minutes  on  the  sections, 
which  are  then  carefully  washed  with  water,  placed  in 
alcohol,  and  finally  stained  again  according  to  the  Gram  or 
the  Gram-Giinther  method. 

*  For  pretty  thin  paraffine  sections  this  time  is  certainly  too  long. 


256  BOTANICAL   MICROTECHNIQUE. 

5.  Staining  Tubercle-Bacilli. 

473.  The  Bacilli  of  tuberculosis  and  of  leprosy  are 
acterized  by  a  peculiar  behavior  with  staining  media  which' 
makes  possible  the  staining  of  them  alone  in  a  mixture  ofj 
Bacteria,  and  therefore  their  certain  distinction  from  othei 
species  of  Bacteria.  Of  the  numerous  methods  recom- 
mended  for  staining  tubercle  Bacilli,  only  the  following, 
due  to  Czaplewski  (I),  need  be  here  referred  to ;  and  I  hav< 
obtained  excellent  results  with  it. 

Cover-glass  preparations  are  treated,  after  fixing,  first  foi 
a  minute  with  carbol-fuchsin  (§  468)  heated  to  boiling.  The] 
are  then  washed  with  the  so-called  Ebner's  fluid  *  until 
hardly  a  trace  of  color  can  be  seen,  are  then  repeatedly 
rinsed  with  pure  alcohol,  and  stained  again  with  a  mixture 
of  three  parts  water  and  one  part  concentrated  alcoholic 
solution  of  methylene  blue.  This  is  then  rinsed  off  with 
water,  and  the  preparation  is  dried  and  mounted,  in  the 
usual  way,  in  balsam. 

If  a  mixture  of  tubercle  Bacilli  and  other  Bacteria,  for 
example,  which  can  easily  be  prepared  by  mixing  pure  cul- 
tures, be  treated  in  this  way,  it  will  be  found  that,  with  the 
exception  of  the  very  rare  \Gipr3.-Bacilli,  only  the  tubercle- 
Bacilli  are  colored  red,  while  all  other  Bacteria  are  blue. 

474.  The  staining  of  tubercle  Bacilli  in  sections  can  be 
accomplished  by  essentially  the  same  methods.  If  one  has 
parafiine  sections  attached  to  the  slide  with  albumen  (§  52), 
they  may  be  heated  in  carbol-fuchsin,  whose  action  for  a 
few  minutes  is  sufficient.  But  if  the  sections  will  not  endure 
heating,  the  fuchsin  solution  must  be  allowed  to  act  for  a 
longer  time  (about  24  hours).  It  is  also  better  to  wash  the 
methylene  blue  from  sections  with  alcohol,  and  to  transfer 
them  to  balsam  through  xylol. 

In  preparations  treated  in  this  way,  only  any  tubercle 
Bacilli  that  may  be  present  are  stained  red ;  all  other  Bac- 
teria, and  the  nuclei  of  the  tissue,  are  blue. 

*  This  consists  of  20  parts  water,  100  parts  alcohol,  .5  part  hydrochloric 
acid,  and  .5  part  sodium  chloride. 


BACTERIA.  257 

6.  Staining  the  Spores  of  Bacteria. 

475.  A  well-differentiated  staining  of  the  spores  of  Bac- 
teria may  be  obtained,  according  to  the  method  proposed  by 
H.  Moeller  (II),  by  plunging  cover-glass  preparations  fixed 
by  heat  or  by  alcohol  in  5^  chromic  acid  "^  for  from  five 
seconds  to  ten  minutes,  then  thoroughly  rinsing  in  water, 
adding  carbol-fuchsin  in  drops,  and  warming  the  whole  in 
the  flame  for  60  seconds,  allowing  it  to  boil  up  once.  The 
carbol-fuchsin  is  then  poured  off,  the  cover-glass  is  plunged 
in  5^  sulphuric  acid  until  it  is  decolorized,  and  again  thor- 
oughly washed  with  water.  An  aqueous  solution  of  methyU 
ene  blue  or  malachite  green  is  then  allowed  to  act  for  30- 
seconds,  and  is  rinsed  off  with  water.  The  preparation  is 
allowed  to  dry  and  mounted  in  balsam.  In  good  prepara- 
tions, the  spores  are  to  be  seen  as  bright  red  spots  within 
the  blue  or  green  bodies  of  the  Bacteria. 

Zinc  chloride  or  chloroi'odide  may  be  used  as  a  mordant,, 
instead  of  chromic  acid,  but  these  commonly  require  a  longer 
time  of  action  than  the  latter. 

7.  Staining  the  Cilia  of  Bacteria. 

476.  For  staining  the  cilia  of  Bacteria,  Trenkmann  (I)  and 
Loeffler  (I)  have  proposed  different  methods.  According  to 
Loeffler's  latest  publication,  the  following  method  is  best 
adapted  for  the  purpose.  The  Bacteria  are  first  spread 
upon  the  carefully  cleaned  cover-glass  (§  459,  note),  and 
fixed  by  being  passed  three  times  through  the  flame,  too 
strong  heating  being  carefully  avoided.  The  mordant  is 
then  placed  on  the  warm  cover-glass.     This  is  best  prepared 

*  In  general,  the  action  of  chromic  acid  for  about  30  seconds  is  sufficient ; 
but  different  species  of  Bacteria  show  great  differences  in  this  respect.  Ac- 
cording to  Moeller,  the  most  favorable  duration  of  its  action  is  :  for  the 
brown  \^oy.2Xo- Bacillus,  30  sec;  for  the  yellow  one,  2  min.;  for  the  while 
one,  10  min.;  for  Bacillus  cyanogenus,  30  sec;  for  the  a.nihra.x  Bacillus,  2 
min.  ;  for  the  tetanus  Bacillus,  2  min.  I  obtained  beautiful  staining  of  the 
spores  of  Bacillus  subtilis  on  allowing  the  chromic  acid  to  act  30  sec.  on 
cover  preparations  fixed  by  heat, 


^58  BOTANICAL   MICROTECHNIQUE. 

"by  mixing  lO  ccm.  of  a  20^  aqueous  tannin  solution,  5  ccm. 
of  a  cold  saturated  solution  of  ferrous  sulphate,  and  i  ccm. 
of  an  aqueous  solution  of  fuchsin.  According  to  the  char- 
acter of  the  Bacteria,  a  few  drops  of  sulphuric  acid  or  of 
caustic  soda*  must  be  added  to  this  mixture.  The  cover- 
glass  holding  it  is  now  warmed  over  the  flame  until  steam  is 
formed  and,  after  half  a  minute  to  a  minute,  the  mordant  is 
rinsed  off  with  distilled  water,  and  the  cover-glass  is  dried 
las  usual.  Then  the  staining  fluid  is  dropped  on  until  the 
Cover-glass  is  wholly  covered  by  it,  the  whole  is  again 
warmed  for  a  minute  until  steam  forms,  and  is  finally  rinsed 
in  a  stream  of  water,  dried,  and  mounted  in  balsam  in  the 
usual  way.  Loefi^er  recommends  as  a  staining-fluid,  a  solu- 
tion of  fuchsin  in  aniline-water,  or  a  mixture  of  100  ccm.  of 
aniline-water,  i  ccm.  of  a  ij^  soda  solution,  and  solid  fuchsin 
in  excess. 

*  For  staining  the  cilia  of  typhus  Bacilli,  22  drops  of  a  \%  aqueous  solu- 
tion of  sodium  hydrate  should  be  added  to  16  ccm.  of  the  above  mordant  ; 
for  Bacillus  sublilis,  28  to  30  drops.  On  the  other  hand,  the  cholera  Bacilli 
require  the  addition  of  ^  to  i  drop  of  a  sulphuric  acid  that  will  just  neutral- 
ize the  same  volume  of  1%  caustic  soda  ;  Bacillus  pyocyaneus,  the  addition  of 
5  to  6  drops  of  the  above  acid ;  Spirillum  rubrum,  9  drops  of  the  same. 
But  the  above  mordant  has  just  the  right  reaction  for  Spirillum  concentri- 
cum.  According  to  the  writer's  experiments,  it  is  also  well  suited  to  Spiril 
ium  Undula  without  further  addition. 


TABLES   FOR   REFERENCE. 


Weights,  Measures,  and  Temperature. 

The  metric  system  is  based  on  the  7neter,  which  was  in- 
tended to  be  one  ten-millionth  of  a  meridian  quadrant  of 
the  earth  in  length. 

The  unit  of  capacity  is  the  liter^  whose  volume  is  that  of 
one  cubic  decimeter  or  looo  ccm. 

The  unit  of  weight  is  the  gram,  which  is  the  weight  of  one 
cubic  centimeter  of  water  at  4°  C. 


The  centigrade  or  Celsius'  thermometer  has  for  its  zero  the 
freezing-point  of  water,  and  for  its  100°  point  the  boiling- 
point  of  water.  One  degree  of  the  scale  is  -^\-^  of  this  in- 
terval. 


Comparison  of  Measures  of  Length. 

English  and  U.  S. 

Metric. 

I  foot 

= 

.3048  meter  =  30.48  cm. 

I       inch 

= 

.0254      " 

=  25.4  mm. 

i       " 

= 

3.175  mm. 

TOTT 

= 

.254     " 

=  254.  ^ 

TTHRT 

= 

.0254  " 

=  25.4-" 

39.37  in. 

= 

I  meter  ^ 

.3937  in. 

= 

I  cm. 

.0394  in. 

= 

I  mm. 

Comparison  of  Measures  of  Capacity. 

English. 

U.S. 

Metnc. 

1  quart,  Imp. 

= 

1.2  qt.,  wine  =  1.135  liter. 

.833  qt.,  " 

= 

I  qt., 

=     .9463    '* 

1  fluid  ounce 

= 

28.38  ccm. 

I     "     dram 

= 

3-55     " 

.8811  quart,  Im] 

p.    =  I. 

0567  qt.,  wine     =  i  liter 

.0352  fl.  oz.,  or 

.2817  fl 

.dr. 

=  I  ccm. 

259 


26o 


TABLES  FOR   REFERENCE. 


Comparison  of  Weights. 


Avoirdupois, 

Apothec. 

Metric. 

I  grain 

= 

.0G48  gram 

I  dram 

= 

.4558  dr. 

= 

1. 7718  " 

2.194  dr. 

= 

I  dram 

= 

3.888   " 

I  ounce 

= 

.9115  oz. 

= 

28.3495  '• 

1.0971  oz. 

= 

I  ounce 

= 

31-1035  " 

lib. 

= 

1. 215  lb. 

= 

453-59 

.8229  lb. 

= 

I  lb. 

= 

373.242   " 

.5643  dr. 

= 

15-432  gr. 

= 

I  gram. 

Comparison  of  Thermometer  Scales. 

r  c.  =  \(t  -  32) "  F.   /'  F.  =  \e  c.  +  32" 


•F. 

•c. 

•F. 

"C. 

»F. 

»C. 

°C. 

0  p 

"C. 

OF. 

»c. 

°F. 

400 

204.5 

130 

54-4 

45 

7.2 

300 

572 

no 

230 

35 

95 

350 

176.7 

120 

48.9 

40 

4-4 

280 

536 

100 

212 

30 

86 

300 

148.9 

IIO 

43-3 

35 

1-7 

260 

500 

95 

203 

25 

77 

280 

137-8 

100 

37-8 

30 

—  I.I 

240 

464 

90 

194 

20 

68 

260 

126.6 

95 

350 

25 

-  3.9 

220 

428 

85 

185 

15 

59 

240 

"5-5 

90 

32.2 

20 

-  6.7 

200 

392 

80 

176 

10 

50 

220 

104.4 

85 

29.4 

15 

-  9-4 

190 

374 

75 

167 

5 

41 

200 

93-3 

80 

26.7 

10 

—  12.2 

180 

356 

70 

158 

0 

32 

190 

87.8 

75 

23.9 

5 

-  15-0 

170 

338 

65 

149 

-  5 

23 

180 

82.2 

70 

21. 1 

0 

-  17.8 

160 

320 

60 

140 

-  10 

14 

170 

76.7 

65 

18.3 

-  5 

—  20.5 

150 

302 

55 

131 

-15 

5 

160 

71. 1 

60 

15-5 

—  10 

-  23-3 

140 

284 

50 

122 

—  20 

-  4 

150 

65-5 

55 

12.8 

-  15 

—  26.1 

130 

266 

45 

113 

-  25 

-  13 

140 

6o.O| 

50 

lO.O 

—  20 

—  28.9 

120 

248 

40 

104 

-  30 

—  22 

Specific  Gravity  and  Percentage  Composition  of 
Solutions. 

The  following  tables  are  based  on  Balances  JiydrometerSy 
a  set  (two)  of  which  is  assumed  to  be  available. 

The  scale  of  the  hydrometer /^r  liquids  lighter  than  water 
has  its  zero-point  at  the  bottom.  This  is  the  point  to  which 
the  instrument  sinks  in  a  10^  solution  of  common  salt.  The 
10°  point  is  that  to  which  it  sinks  in  pure  water.  One  de- 
gree of  the  scale  at  any  part  is  one  tenth  of  this  interval. 

The  hydrometer /^r  liquids  heavier  than  water  has  its  zero 
point  at  the  top  of  the  scale.  This  is  the  point  to  which  it 
sinks  in  pure  water ;  and  the   10°  point  is  that  to  which  it 


TABLES  FOR  REFERENCE. 


26  r 


sinks  in  a  10^  salt  solution.     One  degree  of  the  scale  is  one 
tenth  of  this  interval. 


A  solution  of  a  given  percentage  strength  is  prepared  by 
adding  to  a  number  of  parts  of  the  substance  to  be  dissolved 
equal  to  the  required  percentage,  a  sufficient  quantity  of  the 
solvent  to  make  100  parts  in  all.  Thus,  a  10^  salt  solution 
consists  of  10  parts  of  salt  in  90  parts  of  water. 

For  practical  purposes,  one  cubic  centimeter  of  water  or 
alcohol  may  be  considered  as  one  gram. 

The  figures  in  the  following  tables  refer  to  parts  by  volume 
at  about  60°  F.  Intermediate  values  may  be  obtained  from 
those  given  above  with  sufficient  accuracy  by  interpolation. 


a 

Heavier  than  Water. 

Lighter  than  Water. 

Specific 

% 

^HCl. 

% 

% 

Specific 

% 

% 

e 

Gravity. 

KOH. 

22«   B. 

H,S04. 

HN03. 

Gravity. 

Alcohol. 

NH3. 

"jfl     . 

Italics 

3 
5 
8 

1.022 

2.6 

12.6 

3.8 

4.0 

4J    OJ 

=  % 

1.037 
1.060 

4.5 
7.4 

20.4 
33-6 

5.8 
8.8 

6.3 

10.2 

is 

NH4OH 

26°  B. 

10 

1.075 

9.2 

42.0 

10.8 

12.7 

1. 000 

0. 

0. 

12 

1. 091 

10.9 

50.7 

I3-0 

15.3 

.986 

10. 

3.3 
28.0 

15 

I.II6 

13.8 

64.7 

16.2 

19.4 

.967 

28. 

8.0 

3Q-Q 

17 

1. 134 

15.7 

74.5 

18.5 

22.2 

•954 

38. 

II. 4 

S8.a 

20 

1. 162 

18.6 

89.6 

22.2 

26.3 

.936 

49. 

16.6 

71 S 

22 

1. 180 

20.5 

1 00.0 

24.5 

29.2 

.924 

55. 

20.4 
q2.0' 

25 

1. 210 

23.3 

119.0 

28.4 

33.8 

.907 

62. 

26.3 

27 

1. 231 

25.1 

31.0 

37.0 

.896 

67. 

30.7 

30 

1.263 

28.0 

34.7 

41.5 

.879 

74. 

32 

1.285 

29.8 

37. 

45.0 

.869 

78. 

35 

1.320 

32.7 

41. c 

50.7 

.854 

83. 

37 

1.345 

34.9 

44.4 

55.0 

.844 

87. 

40 

1.383 

37.8 

48.3 

61.7 

.829 

91. 

42 

1. 410 

39-9 

51.2 

67.5 

.820 

94. 

45 

1.453 

43.4 

55.4 

78.4 

.807 

97. 

47 

1.483 

45.8 

58.3 

87.1 

.798 

99. 

50 

1.530 

49.4 

62.5 

lOO.O 

66 

1.842 

lOO.O 

262 


TABLES  FOR  REFERENCE. 


Table  for  Acetic  Acid. 

The  difference  between  the  specific  gravities  of  water  and 
of  glacial  acetic  acid  is  small.  The  mixture  of  the  two  sub- 
stances has  the  peculiarity  that  its  specific  gravity  increases 
up  to  a  certain  point  with  the  addition  of  acid,  and  then 
"decreases  to  that  of  the  pure  acid.  If  the  specific  gravity  is 
above  1.055,  ^"<^  ^^  increased  by  the  addition  of  water,  the 
acid  is  above  78^ ;  if  it  is  decreased  by  the  addition  of 
water,  the  acid  is  below  78^. 


•B. 

Sp.gr. 

%  HC,H,0,. 

»B. 

Sp.  gr. 

j«HC,H,Oa. 

I 
2 

3 
5 

1.0068 
I. 0138 
1.0208 
1.0280 
I.0353 

5.0  — 
9.7  — 

14.6  — 

19.7  — 
25.2  — 

6 

7 
8 

9 
10 

1.0426 
1. 0501 
1.0576 
1.0653 
I.0731 
1.0748 

31.2  — 

37.9  — 
45.6  or  99.1 
55-0  or  95.4 
69.5  or  87.0 

77  to  80 

Table  for  Diluting  Alcohol. 


100  volumes  of  alcohol  of  % 

strength 
of 

90 

85 

80 

75 

70 

65        60 

55 

50 

Alcohol. 

require  addition  of  vols,  water 

85 

6.6 

• 

80 

13.8 

6.8 

75 

21.9 

14.5 

7.2 

70 

31. 1 

23.1 

15.4 

7.6 

$5 

41.5 

33.0 

24.7 

16.4 

8.2 

60 

53.7 

44-5 

35.4 

26.5 

17.6 

8.8 

55 

67.9 

57-9 

48.1 

38.3 

28.6 

19.0 

9.5 

50 

84.7 

73.9 

63.0 

52.4 

41.7 

31.3 

20.5 

10.4 

45 

105.3 

93.3 

81.4 

69.5 

57.8 

46.1 

34.5 

22.9 

11.4 

40 

130.8 

117.3 

104.0 

90.8 

77.6 

64.5 

51.4 

38.5 

25.6 

35 

163.3 

148.0 

132.9 

117.8 

102.8 

87.9 

73.1 

58.3 

43.6 

30 

206.2 

188.6 

171.1 

153-6 

136.4 

118.9 

101.7 

84.5 

67.5 

25 

266.1 

245.2 

224.3 

203.5 

182.8 

162.2 

141. 7 

121. 2 

100.7 

20 

355.8 

329.8 

304.0 

278.3 

252.6 

227.0 

201.4 

176.0 

150.6 

15 

505.3 

471.0 

436.9 

402.8 

268.8 

334.9 

301. 1 

267.3 

233.5 

zo 

804.5 

753.7 

702.9 

652.2 

601.6 

551. 1 

500.6 

450.2 

399.9 

TABLES  FOR  REFERENCE, 


263 


Crystal  Systems. 


Name  of 

Principal  Axes. 

System, 

No. 

Rel.  Length. 

Rel.  Positions. 

Cubic  or 
monometric. 

3 

all  equal. 

at  right-angles. 

Tetragonal 
or  dimetric. 

3 

two  equal, 
third  variable. 

at  right-angles. 

Hexagonal 

4 

three  equal, 
fourth  variable. 

three  at  60"  with  each  other; 
fourth  at  90°  with  these. 

Rhombic  or 
trimelric. 

3 

of  different 
lengths. 

at  right-angles. 

Monoclinic 
or  oblique. 

3 

of  different 
lengths. 

two  at  right-angles, 
third  oblique. 

Triclinic  or 
asymmetric. 

3 

of  different 
lengths. 

all  oblique  to  each  other. 

Isotonic  Coefficient. 

Two  solutions  of  equal  power  to  take  up  water  are  said  by 
De  Vries  to  be  in  isotonic  concentration.  He  terms  a  num- 
ber showing  the  water-absorbing  power  of  a  given  solution, 
as  compared  with  that  of  a  solution  of  saltpeter  of  equal 
strength  taken  as  a  standard,  its  isotonic  coefficient.  For  con- 
venience, the  coefificient  assigned  to  saltpeter  is  3. 

De  Vries  finds  that  compounds  fall  into  six  groups  whose 
coefficients  are  approximately  whole  numbers,  as  follows  : 
I.  Organic   compounds  not   containing  metals,   and    free 
acids ;    e.g.,    Cane-sugar,    tartaric    acid,    citric    acid, 
etc.     2. 

earths  with  one  acid  group  in  the 
Magnesium  sulphate  and  malate.  2. 
earths  with  two  acid   groups  in   the 


II. 


Salts   of  alkaline 

molecule  ;  e.g. 

III.  Salts  of  alkaline 

molecule ;  e.g. 

cium  chloride. 


Magnesium  chloride  and  citrate,  cal- 


264  TABLES  FOR   REFERENCE, 

IV.  Salts  of  alkali-metals  with  one  atom  of  alkali  in  the 

molecule  ;  e.g.,  Potassium  or  sodium  nitrate,  chloride, 

acetate,  etc.  3. 
V.  Salts  of  alkali-metals  with  two  atoms  of  alkali  in  the 

molecule ;  e.g.,  Potassium  sulphate,  oxalate,  tartrate, 

malate,  etc.  4. 
VI.  Salts  of  alkali-metals  with  three  atoms  of  alkali  in  the 

molecule  ;  e.g..  Potassium  citrate.     5. 

Thus  a  solution  of  cane-sugar  has  f  the  water-attracting 
power  of  one  of  saltpeter  of  the  same  strength  ;  and  a  solu- 
tion of  sugar  must  be  |  as  strong  as  one  of  saltpeter  to  pro- 
duce the  same  osmotic  effects. 


LITERATURE. 


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p.  260. 

265 


266  LITERATURE. 

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V.  Ueber  die    Einwirkung   basischer   Stoffe   auf   das   lebende 

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VI.   Neue  Untcrsuchungen   iiber   den  Vorgang  der   Silbcrab- 

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LITER  A  TURE.  267 

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270 


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II.   L'acide  lactique,  excellent  agent  pour  1  etude  des  champig- 

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II.    Ueber   den    mikrochemischen    Nachweis   von  Brucin    und 

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2/4  ^■■r  LITERATURE.  ^^^^^^^ 

Malfatti,   H. — I.    Zur  Chemie  des    Zellkerns.     Ber.    der   naturw.- 
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II.  Beitriige  zur  Kenntniss  der  Nucleine.     Zeitsch.  fiir  physiol 

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II.  Sur  lacallose,  nouvelle  substance  fondamentale  existant  dans 

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IV.  Sur  la  presence  des  composes  pectiques  dans  les  vegetaux. 

lb.     T.  109.     1889.     p.  579. 

V.  Sur  les  reactifs  colorants  des  substances  fondamentales  de  la 

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VI.  Sur  la  substance  intercellulaire.    lb.    T.  no.    p.  295.    (Ref.: 

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VII.  Sur  les  reactifs  jodes  de  la  cellulose.     Bull.  d.  1.  soc.  bot. 

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VIII.    Observations  sur  la  membrane   cellulosique.     Comptes 

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Bedeutung  des  Asparagins  beim  Keimen  der  Samen.  Prings- 
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II.  Ueber    Aufnahme    von   Anilinfarben    in   lebenden    Zellen. 

Untersuchungen  a.   d.   botan.   Instit.  zu  Tiibingen.     Bd.  II. 
p.  179. 

III.  Hesperidin,  ein  Bestandteil  einiger  Hesperideen.     Botan. 

Zeitung.     1874.     p.  529. 

IV.  Beitrage  zur  Kenntniss  der  Oxydationsvorgange  in  lebenden 

Zellen.     Abhandlungen  der  mathem.-phys.  Kl.  d.   K.  Sachs. 
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LITERA  TURE.  277 

Pfeffer. — V.   Osmotische  Untersuchungen.     Leipzig,  1877. 

VI.   Die  Oelkorper  der  Lebermoose,     Flora.     1874.     p.  2. 

Vli.   Low   und    Bokorny's    Silberreduktion    in    Pflanzenzellen. 

lb.    1889.     p.  46. 
— -  VIIL  Zur    Kenntniss    der     Plasmahaut    und    der    Vakuolen. 
AbhandL   d.    math.-phys.   KI,   der  Kgl.  Sachs.  Ges.  d.  Wiss. 
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IX.  Studien    zur   Energetik   der   Pflanzen.      lb.     Bd.    XVIIL 

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2/8  LITERA  TURE. 

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RiCHTER,  K. — I.  Beitrage  zur  genaueren  Kenntniss  der  chemischen 

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Glykoside   und  Alkaloide  in  den  vegetabilischen  Geweben. 

25.    Jahresber.    des    Landes-Realgymnasiums   zu   Stockerau. 

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II.   Beitriige  zur  Histochemie  der  Pflanzen.     Sitzungsbericht  d. 

Akad.  d.  Wiss.  zu  Wien.     1884.     Bd.  89.     Abt.  I.     Mathem.- 

naturw.  Kl. 
ROSTAFINSKI,  J. — Ueber  den  roten  Farbstoff  einiger  Chlorophyceen, 

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II.  Einige   Bemerkungen   iiber  den  Bau   des    Holzes.     Botan. 

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Inaug.-Diss.     Bonn,  1890.      (Ref. :    Zeitschr.   f.    w.  Mikrosk. 
Bd.  VII.     p.  38.) 


LITER  A  TURK.  279 

SCHIMPER. — I.  Ueberdie  Krystallisation  der  eiweissartigen  Substan- 
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II.  Zur    Frage   der   Assimilation    der   Mineralsalze   durch   die 

griine  Pflanze.     Flora.     1890.     p.  207-261. 

III.  Untersuchungen  iiber  die  Chlorophyllkorper  und  die  ihnen 

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SCHOTTLANDER,  P. — I.  Beitrage  zur  Kenntniss  des  Zellkerns  und 

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II.   Beitrage  zur  Kenntniss  der  Chromatophoren.     Pringsheim's 

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II.  Ueber  das  Phycoerythrin.     lb.      1888.     p.  36. 

III.   Ueber  das  Phycophaein.     lb.      1887.     p.  259. 

IV.  Weitere  Beitrage  zur  Kenntniss  des  Phycoerythrins.     lb, 

1888.     p.  305. 
SCHULZE,    E. — I.   Ueber  die    stickstofffreien    Reservestoffe    einiger 
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28o  LITERA  TURE. 

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II.    Ueber  einige   mikroskopisch-chemische  Reaktionen.      lb. 

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SUCHANNEK. — I.  Technische  Notiz  betrefTend  die  Verwendung  des 
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Temme.— I.  Ueber  Schutz-und  Kernholz,  seine  Bildung  und  physio- 
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295.     u.  Bd.  VIII.    p.  34. 


LITERATURE,  28 1 

ViRCHOW,  H.— Ueber  die  Einwirkung  des  Lichtes  auf  Gemische  von 
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Wiesner. — I.  Beobachtungen  iiber  die  Wachsiiberziige  der  Epider- 
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II.  Ueber  die    krystallinische    Beschaflfenheit    der    geformten 

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III.  Note  iiber  das  Verhalten  des  Phloroglucins  und  einigerver- 

wandter  Korper  zur  verholzten  Zellmembran.     Sitzungsber. 
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1878.     p.  60. 
— —  IV.  Ueber  das  Gummiferment.  eln   neues  diastatisches  Enzym, 


282  LITER  A  TURK. 

welches  die  Gummi-  und  Schleimmetamorphose  in  der  Pflanze 

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II.  Ueber  Eisenbakterien.     lb.    1888.     p.  261. 

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WOTHTSCHALL,    E.— Ueber    die    mikrochemischen   Reaktionen    des 

Solanin.     Zeitschr.  f.  w.  Mikrosk.     Bd.  V.     p.  19. 
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Zeitung.     1883.     p.  209. 

II.   Beitrage  zur  Kenntniss  des  Zellkernes  und  der  Sexnalzellen. 

lb.    1887.     Nr.  18  bis  24. 

III.  Ueber  die  Zellen  der  Cyanophyceen.     lb.    1890.    Nr,  1-5. 

IV.  Ueber  das  Wachstum  der  Zellhaut  bei  Wurzelhaaren.    Flora^ 

1891.     p.  467. 

V.  Ueber  die  chemische  Beschaffenheit  von    Cytoplasma  und 

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II.   Beitrage  zur  Morphologie  und  Physiologie  der  Pflanzenzelle^ 

Heft  I.     Tubingen.  1890. 

III.   Idem.     Heft  II.     1891. 

VI.  Idem.     Heft  III.     1893. 

IV.  Eine    einfache   Methode  zur  Sichtbarmachung  des    Torus 

der  Hoftiipfel.     Zeitschrift   f.  w.  Mikroskopie.     Bd.  IV.     p. 
216. 

V.  Botanische  Tinktionsmethoden.    Zeitschr.  f.  w.  Mikroskopie^ 

Bd.  VII.    p.  I. 

VII.   Microchemische  Reactionen  von  Kork  und  Cuticula.      lb. 

Bd.  IX.     p.  58.     1892. 

VIII.  Ueber  die  Fixirung  der  Plasmolyse.     lb.     Bd.  IX.    p.  184. 

1892. 
ZOPF. — I.  Die  Pilztiere  Oder  Schleimpilze.   Schenk's  Handbuch.  Bd. 
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II.   Die  Pilze.     Ibid.     Bd.  IV.     p.  217. 

III.  Ueber  einen  neuen    Inhaltsk(5rper  in   pflanzlichen  Z*»llen. 

Ber.  d.  D.  bot.  Gesellschaft.     1887.     p.  275. 

IV.  Ueber  das  mikrochemische  Verhalten  von  Fettfarbstoffen 


LITEKATURE.  283 

und  Fettfarbstoff-haltigen  Organen.   Zeitschr.  f.  w.   Mikrosk. 

Bd.  VI.     p.  172. 
ZOPF.—  V.   Ueber  Pilzfarbstoffe.     Bot.  Zeitg.    1889.     p.  53. 
VI.    Zur  physiologischen  Deutung  der  Fumariaceen-Behalter. 

Ber.  d.  D.  bot.  Gesellsch,     1891.     p.  107. 
ZWAARDEMACKER,  H. — I.    Flemiiiing's  Safraninfiirbung  unter  Hinzu- 

ziehung  einer  Beize.     Zeitschrift  f.  w.  Mikroskopie.     Bd.  IV. 

p.  212. 


INDEX. 


Abnormal  plasmolysis,  239,  241. 

Abrus  precatorius,  109. 

Absorption  spectrum,  loi,  103,   104, 

105,  106. 
Acanthospheres,  224. 
Acetic  acid,  as  reagent,  60,   63,  71, 

90,  94,  99,  113,  188,  220,  252. 
for  maceration,  6. 

Specific  gravity,  262. 

Achromatic  figures,  194. 
Achyranthes  Verschaffelti,  205. 
Acids,  70,  85. 
Acid  alcohol,  181. 

for  maceration,  6. 

Acidfuchsin,  148,  191,  192,  195,  196, 

197,  202,  205,  207,  213,  218,  246. 
Aconitine,  120, 
Active  albumen,  243,  244, 
ALthalium  septicum,  134,  237. 
Agar-agar  for  attachment,  39. 
Agaricus  armillatus.  Pigment  of,  113. 
Agave,  210. 

Agave  atnericana,  72,  150,  152,  208. 
Aggregation,  241. 
Albumen  for  attachment,  40. 
Alcanna  tinctoria,  71. 
Alcannin,  as  reagent,  71,  74,  89,  90, 

91,  95,  209,  210,  211. 

Alcohol,  as  reagent,  51,  69,  82,  124, 

125. 

for  dehydrating,  12,  32. 

for  fixing,  176,  252,  253. 

Specific  gravity,  261. 

Table  for  diluting,  262. 

Alcohols,  69. 


Aldehydes,  86. 

as  reagents,  131. 

Aleurone,  215. 

Algae,  Study  of,  t,  5. 

Alkaloids,  119. 

Alkyl  thiocarbimides,  81. 

Alliutn,  81,  88. 

Alloxan,  as  reagent,  131. 

Allyl  sulphide,  81. 

Allyl  sulphocyanate,  81. 

Aloe,  Pigment  of  flowers,  103. 

Aloe  verrucosa,  75. 

Altmann's  acid-fuchsin  staining,  195. 

Alum,  181. 

Alum  carmine,  233. 

Alum  cochineal,  184. 

Amido-caproic  acid,  82. 

Amido-compounds,  82. 

Ammonia,    as    reagent,    87,   94,  96, 

113,  122,  123. 
Ammonia-fuchsin,  153. 
Ammonia,  Specific  gravity,  261. 
Ammonio-magnesium  phosphate,  53. 
Ammonium,  57. 
carbonate,  as    reagent,  67,   88, 

117. 

carminate,  182. 

chloride,  as  reagent,  52,  65,  118, 

221. 
molybdate,   as  reagent,  52,  65, 

117. 

oxalate,  6r. 

as  reagent,  66,  167. 

sulphide,  as  reagent,  121. 

vanadate,  as  reagent,  97. 

28s 


286 


INDEX. 


Amorphous  starch,  229. 

Ampelopsis,  63. 

Amphipyrenin,  135,  136. 

Amygdalin,  136. 

Amylodextrine,  229. 

Amyloid,  156. 

Anqiopteris  evecia,  64. 

Anhydrite,  66. 

Aniline  for  dehydrating,  17,  29. 

Aniline  blue,  142,  153,  165,  210,  246, 

247. 

chloride,  as  reagent,  145. 

sulphate,  as    reagent,    86,    145, 

147,  157. 

water,  185. 

solutions,  254. 

Anisic  aldehyde,  as  reagent,  131. 

Anthoceros,  206. 

Anlhochlorin,  107,  108. 

Anihocyanin,  107,  109,  236. 

Antimonic  oxide,  as  reagent,  70. 

Araban,  163. 

Arabanoxylan,  163. 

Arthonia  gregaria,  1 1 2. 

Arthonia-violet,  112. 

Artificial  cells.  Precipitates  in,  243. 

Artificial  precipitates,  242. 

Asaron,  85. 

Asarum  europaum,  85 

Ash-skeletons,  168. 

Asparagin,  51,  82. 

Astrosphere,  19S 

Atropine,  120. 

Attaching  sections  to  slide,  37. 

Attractive  spheres,  198. 

A  vena  orientalis,  21 1. 

Azolla,  243. 

Bacteria,  Fixing  Methods  for,  251. 

by  alcohol,  252. 

by  heat,  251. 

by  lactic  acid,  252. 

Membranes  of.  161. 

Observation  of  living,  250. 

Staining,  253. 

spores,  257. 

cilia,  257. 


Bacteria,  reagent  for  oxygen,  44. 

Bacterio-purpurin,  106. 

Bacterium  termo,  44. 

Balsam  glass,  16. 

Balsam  Tolu,  for  mounting,  224. 

Barium  chloride,  as  reagent,  49,  59, 

62,  70. 
Baryta-water,  as  reagent,  87,  88,  93, 

112. 
Beale's  carmine,  182. 
Beggiatoa,  47. 

Benzol,  as  reagent,  124,  127. 
Berberin,  120. 
Berberis  vulgaris,  120. 
Berlin    blue,    as    reagent,   132,    168, 

172,  190. 

as  stain,  142. 

Bertholletia  excelsa,  74,  219 
Beta,  225. 
Betula  alba,  70. 
Betuloretic  acid,  70. 
Bitter  principles,  99. 
Bohmer's  haematoxylin,  i8l. 
Boletus  edulis,  161. 
Borecole,  237. 
Borodin's  method,  49. 
Borraginacea,  164. 
Bottles  for  reagents,  15. 
Brandt's  reaction,  97. 
Bromine  for  fixing,  176. 
Brucine,  122. 
as  reagent,  51. 

Caffeine  (see  Cofifein),  127. 
Calcium,  57,  66. 

carbonate,  60. 

chloride,  112. 

as  reagent,  70. 

malate,  64. 

nitrate,  as  reagent,  70. 

oxalate,  57,  61,  222. 

phosphate,  64. 

sulphate,  62. 

tartrate,  63. 

Callose,  163,  164. 

Slain  for,  165. 

Callus,  163. 


INDEX. 


287 


Calycin,  99. 

Calycium  chrysocepkalum,  99. 
Campanula  tracheliu?n,  195. 
Canada  balsam,  for    mounting,    11, 

16,  ^o, 

for  sealing,  43. 

Canarin  for  staining,  10. 
Candollea  adnata,  194. 
Cane-sugar,  78, 

as  reagent,  130. 

Canna,  225. 

Canna   Warszewiczii^  205. 
Carbazol,  as  reagent,  145. 
Carbohydrates,  75. 
Carbol-fuchsin,  215,  254,  256,  257. 
Carbon   bisulphide,   as  reagent,   71, 

102. 
Carbonization,  174, 
Carmalum,  183. 
Carmine,  182. 

Beale's,  182. 

P.  Mayer's,  183. 

Carminic  acid,  183. 
Carotin,  loi,  106,  204,  209. 
Caustic  potash  for  clearing,  9. 

for  maceration,  7, 

Caustic  potash,  for  reagent,   59,  63, 

73,  77,  78.  84,  87,  88,  92,  94,  95, 

103,  no,  112,   113,  114,  125,  130, 

137,  151,  209,  217,  233. 

for  swelling,  8. 

Specific  gravity,  261. 

soda,  as  reagent,  139,  141,  164. 

Caulerpa,  168. 

Celloidin  blocks,  Attaching,  36 

Cutting,  36. 

Hardening,  37. 

for  attachment,  28,  38. 

for  imbedding,  36. 

Cell-sap,  Bodies  in,  240. 

Reactions  of,  236. 

Cellulin  grains,  231. 
Cellulose,  138,  139,  149- 

bodies,  231. 

Stains  for,  142. 

Cell-wall,  138. 

Development,  168. 


Cell-wall,  Minute  Structure,  170. 
Centrosome,  198. 
Centrospheres,  198. 

Stain  for,  199, 

Cerasus  lusitanica,  137 
Ceric  acid,  151. 

sulphate,  126. 

ChcBtopteris,  214. 

Chara,  225. 

Characece,  224. 

Chemical  differences  in  walls,  173, 

Chlamydomonas,  215. 

Chloral  carmine,  184. 

Chloral  hydrate,  for  clearing,  9,  10, 

60. 
for   reagent,   71,   90,    193, 

227,  233. 

gelatine,  42. 

Chlorine  for  fixing,  176. 
Chloroform,  as  reagent,  71,  102,  150, 

151. 
Chlorolodide    of    zinc,    as    reagent, 

no,  139,  140,   143,    155,   166,  245, 

246. 
Chlorophyll,  as  reagent,  151. 
Chlorophyll-grains,  136. 

green,  loi. 

yellow,  loi. 

Chloroplastin,  135,  136. 
Chloroplasts,  201. 
Chlororufin,  106. 
Chromatic  figures,  193. 
Chromatin,  134,  136. 

spheres,  191. 

Chromatophores,  201. 

Inclusions  of,  204. 

Minute  Structure,  203. 

Methods  of  study,  5,  201. 

Pigments  of,  100. 

Chrome-yellow,  159. 
Chrom-formic  acid,  178. 
Chromic  acid  for  fixing,  177,  240. 

for  maceration,  7, 

for  reagent,   54,  113,  116, 

125,  257, 

for  swelling,  8. 

Chromic-acid-platinum-chloride.  180. 


288 


INDEX. 


Chromoplasis,  201. 
Chrom-osmic-acetic  acid,  178. 
Chrom-osmic  acid,  235. 
Chrysophanic  acid,  88. 
Chylocladia,  212. 
Cicer  arietinum,  128. 
Cilia,  Stains  for,  214. 

of  Bacteria,  Stains  for,  257. 

Cinchonacece,  210. 
Cinchonamin,  as  reagent,  51. 
Cinnamic  aldehyde,  146. 

as  reagent,  131. 

Citrus  Auraniiutn,  94. 

medica,  58. 

vulgaris,  58. 

Cladophora,  176. 

Cladothrix,  68, 

Clearing  media.  Chemical   9 

Physical,  11. 

Clearing  sections  in  celloidin,  38. 

Clivia  nobilis,  152. 

Closterium,  62,  68. 

Clove-oil  for  clearing,  14,  15,  33. 

Cloves,  84. 

Cochineal,  Czokor's,  184. 

Cocoa-bean,  72,  126. 

Coffee-bean,  73,  94. 

Coffee-tannin,  94. 

Coffein,  as  reagent,  242,  243. 

Colchicine,  122. 

Colchicum  officinale,  122. 

CoUus,  249. 

Collodion,  for  attachment,  37,  39. 

Coloring  matters,  100. 

Combretacea,  210. 

Comparison  of  therixLometers,  260. 

of  weights  and.  measures,  259. 

Concentration  of  a'.cohol,  13,  28. 
Copt/ervacea,  68. 
Congo  red,  143,  169. 
Coniferce,  92,  142. 
Coniferin,  92,  144    146. 
Conjugate,  Sheaths  of,  157. 
Constitution  of  resting  nucleus,  191. 
Convolvulus  tricolor,  205. 
Copper   sulphate,    as    reagent,    130, 
137. 


Corallin,  as  reagent,  155,  104. 

Cordiacece,  2 ID. 

Cork,  149,  152. 

Corrosive  sublimate  for  fixing,  179^ 

213,  218. 
Corydalin,  122. 
Cosmarium,  206. 
Cover-glass  preparations,  251. 
Creosote  for  clearing,  29. 
Crocin,  96. 
Crocus  vernus,  242. 
Cruciferce,  95,  137. 
Crystalloids,  Staining,  195. 
Crystals,  Observation  of,  43. 
Crystals    of     ammonio- magnesium 

phosphate,  53. 

asparagin,  51,  69,  83 

berberin,  121. 

calcium  oxalate,  58,  60. 

sulphate,  66. 

tartrate,  63,  7 

dulcite,  69. 

gypsum,  62. 

hesperidin,  94. 

piperine,  125. 

protein-grains,  221. 

saltpeter,  51,  69,  83. 

silver  chloride,  48. 

sulphur,  47. 

,  Preparation  of,  43. 

Crystal  Systems,  263. 
Cucurbit acea:,  246,  249. 
Cucurbita  Pepo,  168,  248. 
Culture  slide  for  Algae,  3. 
Cuprammonia,  as  reagent,  139,  140,. 

155,  157,  162. 
Cuprammonia  for  swelling,  8. 
Cupric  acetate,  as  reagent,  90,  115. 

sulphate,  as  reagent,  77,  78. 

Curcuma  a  mat  a,  107. 

Curcumin,  107. 

Cuticle,  148,  150,  152. 

Cyan  in,    46,   72,  90,    152,    154,    209, 

211,  214,  238. 
Cyanophilous  nuclei,  191. 
Cyanophycece,  109,  232. 
Pigments  of,  104. 


INDEX. 


2% 


Cyanophycin-grains,  233. 
Cycas  circinalis,  58. 
Cynoglossum,  164. 
Cystoliths,  61. 
Cytisine,  123, 
Cytisus  Laburnum,  123. 
Cytoplasm,  Bodies  in,  240. 
Cytoplastin,  135,  136. 
Czaplewski's    stain   for   tubercle-Ba- 
cilli, 256. 

Dahlia,  stain,  189. 

Dahlia  variabilis,  79,  82,  83,  86. 

Dammar  lac  for  mounting,  18 

Datisca  cannabina,  93. 

Datiscin,  93. 

Dauctis  Carofa,  loi,  203. 

Dehydrating  vessel,  Schulze's,  12. 

Dehydration,  11. 

by  alcohol,  12. 

by  drying,  17. 

Klercker's  method,  12. 

Overton's  method,  13. 

Delafield's  haematoxylin,  180. 

Delicate  objects,  To  mount,  15,  18. 

Dermatosomes,  174. 

DesmantJiMS  plenus,  234. 

Desmidiacea,  17,  157,  160. 

Development  of  cell-wall,  168. 

Dextrine,  80, 

Dextrose,  77. 

Diastase,  229. 

DiatomacecEy  Pigments  of,  105. 

Diatomin,  105. 

Dicranochcele  reniformis,  207. 

Digestive  fluids  for  chromatin,  192. 

Dioncca,  222,. 

Diphenylamine  as  reagent,    50,    69, 

83. 
Discocrystals,  229. 
Dishes  for  staining,  24. 
Double  staining,  147. 
Draccena,  210. 

Draining  boxes  for  washing,  22. 
Drosera  dichotoma,  223. 

rotundifolia,  241. 

Dulcite,  69. 


Eau  de  Javelle,  for  bleaching,  6. 

for  clearing,  10. 

for  reagent,  no,  145, 

152,  228    233. 

Ehrlich's  solutions,  254. 

Elaioplasts,  209. 

Ellagic  acid,  86. 

Ely??t  us  g  iga  n  teus,  211. 

Emodin,   87. 

Emulsin,  136. 

Eosin,  153,  154,  165,  185,  199,  216, 
218,  233,  246. 

Eosin-haematoxylin,   199. 

Epiphylluni,  222. 

Equisettwi  arvense,  204. 

hiemale,  54. 

Erysipkece,  231. 

Erythrophilous  nuclei,  191. 

Eternod's  apparatus,  25. 

Ethereal  oils,  89. 

Eugenol,  84. 

Euglena  acus,  230. 

Ehrenbergii,  230. 

Spirogyra,  230. 

Euphorbia,  225. 

caput-m^dusce,  64. 

Evonynius  japonicus,  69. 

Exclusion  of  Bacteria  from  cult- 
ure, 4. 

Eye-spot,  209. 

Fats  and  fatty  oils,  71. 

Fehling's  solution,  77,  78,  92. 

Ferments,  136. 

Ferric  acetate,  as  reagent,  115. 

chloride,  as  reagent,  91,  93,  94, 

95,  98,  115,  132,  143. 
Ferrous  sulphate,  as  reagent,  45,  95, 

98,  115,  2ig. 
Fibrosin-bodies,  231. 
Ficus  elastica,  61,  62. 
Fixing-fiuids,  Removal  of,  22. 
Fixing,  Methods  for,  20,  21,  27. 
Fixing-methods     for     cell-contents, 

176. 
Flemming's  fixing-fluid,  178. 
FloridecE,  230 


290 


INDEX. 


./'loridece,  Pigments  of,  103. 
Floridean  starch,  230. 
fluids  for  study  of  living  cells,  4. 
Fceniculum  officitiale,  162. 
Frangulin,  93. 

Fuchsin,  147,  188,  191,  203,  204,  258. 
Fucus,  176. 
Fundamental  mass  of  protein-grains, 

216. 
Fungus-cellulose,  160,  232. 
Fungus-gamboge,  91. 
Fungi,  Study  of,  i,  5. 
Funkia,  210. 

Gaertneracea,  210. 

Galactose,  163. 

Garlic  oil,  81. 

Gelatinized  walls,  154. 

Gelatinous  sheaths  of  Conjugatce,  157. 

Gentian  violet,    148,    153,    185,    186, 

255. 
Globoids,  219. 
Glceocapsa,  I  ID. 
Gloeocapsin,  no. 
Glucose,  77. 
Glucosides,  92. 
Glycerine  for  clearing,  11. 

for  dehydrating,  13. 

for  mounting,  41. 

Glycerine     and    chrome    alum     for 

mounting,  41. 
Glycerine-gelatine  for  mounting,  41, 

42. 
Glycogen,  80. 
Gold-chloride,  as    reagent,    81,    122, 

126,  127. 
Gold-size  for  sealing,  43. 
Gonium  pectorale  214. 
Graminea:,  54,  210. 
Gram-GUnther  method,  255, 
Gram's  method  for  staining,  185,  255. 
Grana  of  chromatophores,  204. 
Granula,  213. 
Gratiola  officinalis,  222. 
Grenacher's  borax-carmine,  182. 

haematoxylin,  180, 

Growth  of  cell-wall,  168. 


Guiacum  officinale,  5 J 

Gums,  154. 

Gypsum,  59,  62,  63,  64,  65,  66. 

Haematein,  181. 
Haematochrome,  106. 
Hcematococcus,  106. 
Haematoxylin,    142,     153,    180,    197^ 

208. 
Hanging  drop  culture,  2. 
Hebeclinium  7nacrophyllum,  63. 
Hedera,  205. 
Helianthus  annuus,  74. 
Helichrysin,  109. 
Helichrysiim,  109. 
Hemicelluloses,  161. 
HepaticcB,  210. 
Hesperidin,   93. 
Higher  plants.  Study  of,  5. 
Hoffmann's  reagent,  130. 
Hofmann's  blue,  224,  247. 

violet,  245,  247. 

Humulus,   2^9. 

Hydrocarbons,   88. 

Hydrocellulose,  142, 

Hydrochloric  acid,  48. 

as  reagent,  58,  65,"  67,  84, 

86,  90,  no,  113,  121,  126,  127,  137, 

194. 

Specific  gravity,  261. 

Hydrofluoric  acid,  as  reagent,  53,  55. 
Hydrogen  peroxide,  45,  117,  178. 
Hydrolysis,  139,  162,  163. 
Hydroxylamine,  as  reagent,  147, 

Imbedded     objects.  Attachment    to 

carrier,  35. 
Imbedding  in  celloidin,  35. 

paraffine,   31. 

Impatiens  Balsamina,  156. 

parvijlora,  148. 

Inclusions    of  chromatophores,  204. 

of  nucleus,   194. 

India-ink,  Use  of,  158. 
Indol,  as  reagent,  145. 
Inorganic  Compounds,  44. 
Intercellular  substance,  166. 


INDEX. 


291 


Inulin,  78. 

Invert-sugar,  78. 

Iodine,  as  reagent,  155,  227. 

for  fixing,  27,  176. 

for  removing  sublimate,  179. 

Iodine  in  sea-water  for  fixing,  212. 

Iodine  and  potassium  iodide  for  fix- 
ing, 214,  224. 

as  reagent,   80, 

102,  103,  106,  120,  122,  123,  128, 
129,  185,  186,  233. 

and  sulphuric  acid,  as  reagent, 

no,  139,  140,  143. 

Iodine-calcium  chloride,  as  reagent, 
141. 

Iodine-green,  188.  202. 

Iodine-phosphoric  acid,  as  reagent, 
141. 

Iridescent  plates  of  Algae,  212. 

Iridous  chloride,  as  reagent,  124. 

Iron,  68. 

Isotonic  coefficient,  238,  263. 

Javelle  water,  see  Eau  de  Javelle. 
Juglans  regia,  87. 
Juglon,  87. 

Karyokinetic  figures,  185,  187,  192, 
Kinoplasm,  194. 

Lactic  acid  for  dried  plants,  5. 

for  fixing,  252. 

Lamellation  of  wall,  170, 

Lathrea  squamaria,  225. 

Lead  acetate  as   reagent,  93,  94,  98, 

107,  109. 
LeguminoscE ,  162. 
Lenzites  sepiaria,  92. 
Lepidium,  169. 
Lepra- Bacillus,  256. 
Leptoniihts  lacteus,  23 1. 
Leptophrys  vorax,  230. 
Leptothrix  ochracea,  69. 
Leucin,  82. 
Leucoplasts,  201. 
Leucosomes,  205. 
Lichen-pigments,  no,  in. 


Life  reagent,  244. 
Lignic  acids,  144. 
Lignified  walls,  143. 

Rea(!tions  of,  145. 

Lignin,   143. 

Liliutn  Martagon,   ig8,  199. 

Lime  water,  as  reagent,  87,  88,  93, 

96,  113. 
Linin,  135,  136. 
Lipochromes,  106. 
Lipocyanin,  106. 
Lithospermum,  164. 
Live-staining,  119,  189,  224,  235. 
Living  Bacteria,  Study  of,  250. 
Living  tissues,  Staining,  19,  119. 
Loeffler's  blue  stain,  253. 
Loevv-Bokorny  reagent,  244. 
Lophospermum  scandenSy  195. 
Lupinus  luteus,  162. 

Maceration,  6, 
Madder  dye,  95. 
Magnesium,  67, 

oxalate,  67. 

phosphate,  67. 

sulphate,  52,  65. 

Mandelin's  reaction,  97. 
Mannose,  162. 
Marattiacece,  63. 
Masked  iron,  68. 
Maskenlack  for  sealing,  43. 
Mass-staining,  182. 
Measures  of  capacity,  259. 

length,  259. 

Melampyrite,  69. 
Melampyrum  arvense,  194. 
Membranes  of  Bacteria,  161. 
Mercuric   chloride,  as   reagent,  122, 
128. 

iodide,  57. 

for  swelling,  8. 

nitrate,  as  reagent,  129. 

Mesocarpus,  234. 
Metadiamidobenzol,  as  reagent,  86, 

157. 
Metaxin,  135,  136. 
Methylal,  as  reagent,  124. 


29^ 


INDEX. 


Methyl  alchol,  171. 

blue,    as    stain,    142,    153,    188, 

190. 

as  reagent,  1 19. 

Methylene  blue,   166,   168,  173,  191, 

192,  224,  235,  248,  256. 

Loeffler's,  253. 

Methyl  green,  188,  190. 

orange,  as  reagent,  236,  238. 

Methyl  violet,  1S9,  247,  254. 
Micrasterias  rotata,  62. 
Microcosmic  salt,  as  reagent,  67. 
Microsomes,  212. 
Microtome  knife,  31. 
Microtomes,  29,  30. 
Microtome  technique,  29. 
Middle  lamella,  6,  167. 
Millon's  reagent,  85,  129,  137. 
Mimosa     pudica,    Glucoside     from, 

98. 
Mimulus  Tillingi,  195. 
Minute  structure  of  cell-wall,  170. 
Moist  chamber,  2. 
Mordant  for  cilia  of  Bacteria,  258. 

spores  of  Bacteria,  257. 

Morphine,  123. 

Mounting  delicate  objects,  15. 

in  air,  43. 

in  balsam,  16. 

with  alcohol,  12. 

drying,  17. 

phenol,  17. 

Mucus-globules,  232. 
Musa  paradisiaca,  58. 
Mustard  oils,  81. 
Myrosin,  81,  137. 

a-Naphtol,  as  reagent,  76,  79,   145. 
Narcelne,  124. 
Narcotine,  124. 
Neottia  nidus-avis^  204. 
Nephroma  lusitanica,  87. 
Nerium,  171,  172,  173. 
Nessler's  reagent,  57. 
Nickel  sulphate,  as  reagent,  50. 
Nicotine,  128. 


Nigrosin,  189. 
Nitella,  190,  224,  225. 
Nitric  acid,  50. 

for  fixing,  213. 

for  maceration,6. 

for  reagent,  52,  54,  65,  85, 

86,  96,  103,  112,  113,  121,  122,  129, 

156. 

Specific  gravity,  261. 

Nostoc,  233. 

Nucin,  87. 

Nuclear  divisions,  Fluid  for,  4. 

Nuclear  membrane,  192, 

Nucleic  acids,  133. 

Nuclein,  234. 

— '-  Artificial,  134. 

Nucleins,  133. 

Nucleolus,  191. 

Nucleus,  Constituents  of,  175. 

Inclusions  of,  194. 

Oil-bodies,  210. 

Oil-drops,  208. 

Oil-formers,  2og, 

Oil  of  bergamot  for  clearing,  39. 

Oils,  Ethereal,  89. 

To  distinguish,  90. 

Fatty,  71. 

for  clearing,  14. 

Ononis  spiuosa,  90. 

Opium  alkaloids,  123. 

Oplismenus  imbecillus,  21 1. 

Orange,  stain,  186. 

Orcin,    as  reagent,    79,   84,   86,   136, 

137.  145,  157- 
Organic  compounds,  69. 
Ornithogalum,  2 ID. 
Orseillin,  199. 
Oscillatoria,  104. 
Osmic  acid,  as  reagent,  27,   72,  90, 

117,   152,  178,    211,  212,   213,   2I4» 

215,  219.  239. 
Overstaining,  26. 
Oxalic  acid,  62,  70. 
Oxalic  acid  for  maceration,  6. 
Oxygen,  44. 
Oxynaphthoquinone,  87. 


INDEX, 


^93 


Pceonia,  156,  217,  219. 

Fallacious  chloride,  as  reagent,  124. 

nitrate,  as  reagent,  81. 

Pancreatin,  as  reagent,  133. 
Pandorina,  215. 
PanicecBy  67. 

Papaver  somniferum,  123. 

Paracarmine,  183. 

Paraffine  blocks,  To  attach,  35. 

To  preserve,  35. 

for  imbedding,  32. 

oven,  34. 

Paragalactan,  162. 

Paragalactan-like  substances,  161. 

Paralinin,  135,  136. 

Paramylum,  230. 

Paris  quadrifolia,  162. 

Paspalum  elegans,  82. 

Passijlora  ccerulea,  ill. 

Paxilhis  atrotomentosus,  Pigment  of, 

113- 
Pectic  acid,  167. 

substances,  166. 

Pellicle  of  protein  grains,  217. 

Penicillium,  48. 

Pepsin,  as  reagent,  133,  134,  252. 

Peridinecc,  Pigment  of,  105. 

Peridinin,  105. 

Permanent  preparations,  40. 

PeronosporecE,  164. 

Peziza,  187. 

Phacus  parvula,  230. 

PhcEophycecB,  2y:i. 

Pigments  of,  104. 

Phaeophycean  starch,  230. 
Phellonic  acid,  149. 
Phenol,  for  clearing,  10,  60. 

for  dehydrating,  17. 

for  reagent,  71,  i45i  146. 

Phenols,  84. 

Phenosafranin,  166,  168. 
Phloicnic  acid,  149. 
Phloridzin,  95. 
Phloroglucin,  84,  119. 

as  reagent,  80,  86,  144, 145.  I47» 

157. 
Phoenix  dactylifera,  162. 


Phosphoric  acid.  52. 
Phospho-molybdic  acid,  as  reagent^ 

119,  120,  123. 

-tungstic  acid,  as  reagent,  128. 

Photophore,  25, 

Photoxylin  for  imbedding,  37. 

Phycocyanin,  105. 

Phycoerythrin,  104. 

Phycophsein,  104. 

Phycopyrrin,  105. 

Pkyllosiphonacea,  231. 

Physcia  parietina,  88. 

Physodes,  214. 

Phytelephas,  162,  174. 

Phytophysa  Treubii,  231. 

Picric  acid,   for  differentiating,   i82> 

188. 
for    fixing,   177,   189,    207» 

210,  216,  235. 

for  reagent,  123. 

Picrocarmine,  183,  255. 
Picro-nigrosin,  189,  216. 
Picro-sulphuric  acid,  177,  235. 
Pigments,  100. 

dissolved  in  cell-sap,  107. 

in  oils,  107. 

Fatty,  106. 

in  the  cell-wall,  108,  109. 

of  Aloe  flowers,  103. 

Chromatophores,  100. 

CyanophycecE,  104. 

DiatomacecE  y  105. 

FloridecE,  103. 

lichens,  no,  in. 

Peridinece,  105. 

•  Pha:ophyce(E,  104. 

on  the  cell-wall,  112. 

Piperacea,  124. 

Piperine,  124. 

Pirus  Mains,  95, 

Pistia  Stratiotcs,  235. 

Plant-mucilages,  154. 

Plasma-membranes,  238. 

Plasmolysis,  238,  243. 

Plastin,  134. 

Plastoids,  223, 

Platinum  chloride,  for  fixing,  180. 


294 


INDEX. 


Platinum    chloride,  for  reagent,   56, 
81,  128. 

-osmic-acetic  acid,  for  fix- 
ing, 180. 

Pleochroism,  204. 

Pleurotccnium  Trahecula,  159. 

Plugge's  reagent,  130. 

Podocarpus  elongatus,  173. 

Podosphara  Oxyacanthce,  231, 

Polarized  light,  60, 65, 73, 88,  220, 226. 

Polygonacea,  88. 

Polypodiiim  irreoides,  222,  240. 

Polyporus  hispidus,  91. 

Potamogeton,  ll\. 

Potassic-mercuric    chloride,    as    re- 
agent, 122. 

iodide,    as    reagent,     122, 

123,  12S. 

Potassium,  56. 

acetate,  as  reagent,  70. 

bichromate  for  antiseptic,  4. 

for  differentiating, 

1S2,  192,  197. 

for  reagent,  116,  123,  219. 

-bismuth  iodide,  as  reagent,  123. 

-calcium  iodide,  as  reagent,  123. 

carbonate,  as  reagent,  86. 

caryophyllate,  84. 

chlorate  for  maceration,  6. 

chromate,  4,  122. 

ferrocyanide,    as    reagent     68, 

132,  135,  172,  igo,  192. 

hydrate.     See  Caustic  potash. 

iodide,  45,  57. 

myronate,  81,  95. 

nitrate,  51. 

as      reagent.      See     Salt- 
peter. 

oxalate.  Acid,  as  reagent,  70. 

permanganate   for   differentiat- 
ing, 200. 

platinum  chloride,  56. 

sulphate,  49,  50. 

sulphocyanide,  as  reagent,  68, 

123. 
Pritnulacea,  156, 
Protelds,  128. 


Proteids,  Reactions  of,  129. 
Protein    crystalloids,   194,   205,   217, 
222. 

grains,  215. 

Proteosomes,  244. 
j  Protoplasm  and  cell-sap,  174. 
Protoplasm,  Reactions  of,  237. 
Protoplasmic  Connections,  245. 
Prunus  Lauro-cerasus,  136. 
Pulmonaria  officinalis,  236. 
Pulverization  methods,  174. 
Pyrenin,  135,  136. 
Pyrenoids,  206. 
Pyroligneous  acid,  180. 

Quercus  Suber,  149. 
Quinones,  87. 

Raphides,  58,  60. 
Raspail's  reagent,  138,  224. 
Reactions  of  cell-sap,  236. 

of  protoplasm,  237 

Reagent-bottles,  1=;. 

Peniijia  purdieana,  51. 

Removing  air  from  tissues,  5. 

Replacement  of  alcohol,  14. 

Resedacecc,  137. 

Reserve-cellulose,  162. 

Resin,  211. 

Resins,  90. 

Resorcin,  as  reagent,  86,  145. 

Resting  nucleus.  Recognition  of,  190. 

Retinic  acids,  91,  92 

Rhabdoids,  223. 

Rhamnus  frangula,  87,  93. 

Rhodospermin,  223. 

Riciitus,  117,  118,  130,  216,  218,  219. 

Rochelle  salt,  as  reagent,  77. 

Ruberythric  acid,  95. 

Ruhiacea,  210, 

Rubia  iinciorutn,  95. 

Rutin,  96. 

Saccharose,  78. 

Saffron-yellow,  96. 

Safranin,  148,  152,  185,  186,  207. 

Salicin,  96. 

Salicylic  aldehyde,  146. 


INDEX. 


29s 


Salicylic  aldehyde  for  fixing,  202. 

for  reagent,  131,  132. 

Salt,  238. 

Saltpeter,  5,  240,  263. 

Sapindacece,  210. 

Saponaria  officinalis,  230. 

Saponification  of  fats,  73. 

Saponin,  96. 

SapotacecE,  210. 

Sap  rolegn  ia  cecE,  231. 

Schulze's  dehydrating  vessel,  12. 

macerating  mixture,  6,  151. 

settling  cylinder,  16, 

Schweizer's  reagent,  140. 

Sculpturing  of  wall,  170. 

Scytonefna,  no,  233. 

Scytonemin,  no. 

Sealing  media,  43. 

Sec  ale  cereale,  148. 

Section-finder,  25. 

Selenic  acid,  as  reagent,  122. 

Seminin,  162. 

Seminose,  162. 

Setaria  viridis,  67. 

Settling  cylinder,  Schulze's,  16. 

Sieve-plates,  246,  248. 

Sieve-tubes,  Callus  of,  163,  165. 

Contents  of,  248. 

Silica  skeletons,  54,  168. 

Silicic  acid,  53. 

Silvering,  173,  226. 

Silver  nitrate,  as  reagent,  48,  81,  85, 

173. 
Silver-solution,  244. 
Sinapine,  125. 
Skatol,  as  reagent,  145. 
Small  objects.  Fixing  and  Staining,27. 
Smilax,  225. 
Soda  solution,  165. 
Sodium,  56. 

carbonate,  as  reagent,  113. 

carminate,  183. 

hydrate,  see  Caustic  soda. 

metatungstate,  as  reagent,  122. 

phosphate,  as  reagent,  67,  217, 

218,  220. 
selenate,  as  reagent,  97,  124. 


235. 


Sodium  silico-fluoride,  55. 

tungstate,  as  reagent,  117. 

uranyl  acetate,  56. 

Solanin,  97. 

Solanum  tuberosuju,  202,   222. 

Soluble  blue  extra  6B,  165. 

Soluble  starch,  229. 

Solutions,  Percentage   Composition^ 

260. 

Preparation  of,  261. 

Solvents  for  fats,  71. 

Sorbus  aucupaHa,  204. 

Specific  gravity  of  solutions,  260. 

Spergula  vulgaris,  99. 

Spergulin,  99. 

Sphaerocrystals,    63,   64,  65,   73,    74,. 

79,  82,  85,  93. 
Spirogyra,  18,  45,  115.  118,  131,  132,. 

206,  238,  240. 
Spores  of  Bacteria,  Staining,  257. 
Staining  attached  sections,  38. 

in  mass,  24,  182. 

intra  vitam,  119,  189,  224, 

living  tissues,  19. 

Methods  for,  20,  24,  27. 

methods  for  cell-contents, 

sections,  25. 

Stains  for  cellulose,  ^42,  143. 

cuticle  and  cork,  152, 

lignified  walls,  147,  148. 

Stapelia  picta,  52,  53,  64. 
Starch-grains,  225. 

Medium  for,  19. 

Starch,  Recognition  of,  227. 
Starch  skeletons,  229. 
Staurastrum  bicorne,  159. 
Stender  dishes,  24. 
Stratification  of  cell-wall,  170. 

of  starch-grains,  226. 

Striation  of  cell-wall,  170. 

Strontium  nitrate,  as  reagent.  49. 

Strychnine,  125. 

Strychnos  nux-vomica,  122,  126. 

Suberic  acid,  149. 

Suberin,  148,  150. 

Suberized  membranes,  75,   148,  150, 

152. 


[80. 


154. 


296 


INDEX. 


Sulphur,  47. 

compounds,  81. 

Sulphuric  acid,  49. 

for  maceration,  7. 

for  reagent,  54,  59,  62,  64, 

65,  66,  67,  68,  70,  79,  81,  84,  86, 
88,  91,  92,  96,  97,  98,  99,  102,  103, 
105,  io6,  107,  112,  113,  120,  122, 
124,  127,  130,  140,  164,  246. 

for  swelling,  8. 

Specific  gravity,  261. 

Sulphurous  acid  for  washing,  22, 177. 

Swelling,  8. 

Syringa  vulgaris,  98. 

Syringin,  98. 

Tables  for  reference,  259.     ' 

Tannic  acid,  242. 

Tannin,  as  reagent,  45,  143. 

Tannins,  114,  242. 

Tannin-vesicles,  234. 

Tartaric  acid,  as  reagent,  70,  119. 

Terpenes,  90. 

Thallin  sulphate,  as  reagent,  49,  145, 

146. 
Theine,  127. 
Thelephora,  91,  112. 
Thelephoric  acid,  112. 
Theobromine,  126. 
Thermometer  scales,  260. 
Tholuidendiamine,  as  reagent,  145. 
Thymol,  as  reagent,  76,  79,  86,  145, 

157. 
Titanic  acid,  as  reagent,  124, 
Tradescantia,  200. 

albijlora,  214. 

discolor,  58,  205,  206,  239,  243. 

virginica,  46,  189. 

Trametes  cinnabarina,  91,  113. 
Trianea  bogotensis,  46. 
Tropaolacea,  137, 
Tropceolitm  ma  jus,  156. 
Trypsin,  192. 

Tubercle-bacilli,  Staining,  256. 
Tulipa  suaveolens,  242. 
TurnbuU's  blue    169. 
Tyrosin,  85. 


Unequal  water-content  of  cell-walls, 
171. 

Unverdorben  -  Franchimont  reac- 
tion, 90. 

Uranyl-acetate,  as  reagent,  56,  67,  70. 

Uranyl-magnesium  acetate,  as  re- 
agent, 56. 

Urceolaria  ocellata,  112. 

Urceolaria-red,  112. 

Urticales,  164. 

Vanda  furva,  222. 
Vanilla,  86. 

planifolia,  209. 

Vanillin,  86,  144,  146. 

as  reagent,  84,  131. 

Venetian    turpentine  for  mounting, 

18. 
Veratrine,  127. 
Veratrum  album,  127. 
Vessel  for  staining  sections,  26. 
Vesuvin,  165. 
Vicia  Faba,  46,  187. 
Vinca,  172,  173. 
ViolacecE,  137. 
Vitis  Labrusca,  63. 
vinifera,  220,  222. 

Washing,  22,  27. 

apparatus,  23,  27. 

Wax,  74. 

Wax  feet  for  cover-glass,  i. 
Weights,  Table  of,  260. 
Willows,  96. 
Wood-gum,  144. 
Wound-gum,  157. 

Xanthein,  107. 
Xanthin,  103,  106,  209. 
Xanthine,  128. 
Xantho-proteic  acid,  129. 
Xanthotrametin,  113, 
Xylol  for  clearing,  14. 
for  imbedding,  33. 

Zinc  chlorine,  as  reagent,  231. 

sulphate,  as  mordant,  199, 

Zygnema,  115,  176,  206,  235. 
Zygnemacece,  157,  158,  234. 


152808  /^ 


